Abstract:

The invention provides methods for predicting whether an ovarian cancer
patient's tumor will be resistant to chemotherapy. The invention also
provides methods for monitoring the effectiveness of treatment,
particularly a chemotherapeutic treatment, in a patient treated for
ovarian cancer. The invention further provides methods for treating
ovarian cancer, by reducing chemotherapeutic drug resistance in said
cells. In addition, the invention provides methods of screening compounds
to identify tumor cell growth inhibitors in tumor cells resistant to
conventional chemotherapeutic treatment regimes.

Claims:

1. A method of assessing whether an ovarian cancer patient's tumor is
resistant to cisplatin comprising the steps of:(a) measuring a gene
expression level of Calpain 2 in:(i) an ovarian cancer tumor sample taken
from the patient, and(ii) a cisplatin responsive ovarian tumor sample;(b)
comparing the expression level of said gene in the ovarian cancer tumor
sample taken from the patient and the cisplatin responsive ovarian tumor
sample; and(c) determining that the ovarian cancer patient's tumor is
resistant to cisplatin when the expression level of said gene in the
ovarian cancer tumor sample taken from the patient is at least 1.2-fold
greater than the expression level of said gene in the cisplatin
responsive ovarian tumor sample.

2. A method for assessing disease progression of ovarian cancer in a
patient, comprising the steps of:(a) measuring a gene expression level of
Calpain 2 in an ovarian cancer tumor sample taken from the patient;(b)
repeating step (a) using an ovarian cancer tumor sample obtained from the
patient at a subsequent time;(c) comparing the expression level of said
at least one gene from step (a) with the gene expression level from step
(b); and(d) determining that the cancer has progressed when the
expression level measured in step (b) is not less than the expression
level measured in step (a).

5. The method of claim 1 or 2, wherein said expression levels are measured
by RT-PCR, and wherein said expression levels are measured relative to
expression levels of a control gene.

6. The method of claim 5, wherein the control gene is 18S RNA gene.

7. A method of assessing treatment efficacy in an ovarian cancer patient
comprising the steps of:(a) assaying an ovarian cancer tumor sample from
the patient for an expression level of Calpain 2;(b) repeating step (a)
at a subsequent time during treatment of the subject for ovarian
cancer;(c) comparing the expression level of said gene from step (a) with
the gene expression level from step (b); and(d) determining that the
treatment is efficacious when expression level of said gene in step (b)
is less than the expression level in step (a).

Description:

[0001]This application is a divisional of U.S. application Ser. No.
11/026,734, filed Dec. 30, 2004, which claims priority to U.S.
provisional application Ser. No. 60/533,505 filed Dec. 31, 2003, the
disclosure of which is incorporated by reference herein.

FIELD OF THE INVENTION

[0002]The invention relates to methods for predicting whether an ovarian
cancer patient will be resistant to chemotherapy and methods for
determining whether an individual patient has colon cancer. The invention
also relates to methods for monitoring the effectiveness of therapy in a
patient treated for ovarian cancer. The invention further relates to
methods for treating ovarian cancer and colon cancer. In addition, the
invention relates to methods of screening for compounds that can inhibit
growth of tumor cells, particularly ovarian cancer cells or colon cancer
cells. The invention also relates to methods for reducing or inhibiting
resistance to chemotherapeutic drug treatment or therapy, particularly in
ovarian cancer cells that are resistant to conventional chemotherapeutic
treatment regimes.

BACKGROUND OF THE INVENTION

[0003]Ovarian cancer is the most lethal of gynecological malignancies with
a mortality rate of 60%. The five-year survival rates for the various
clinical stages of the disease are as follows: Stage I>90%, Stage
II=80%, Stage III=20% and Stage IV=10%; there is a significant drop in
the survival rates at later stages of the disease. Standard-of-care
treatment for advanced stages of the disease includes cytoreductive
surgery followed by chemotherapy.

[0004]For most patients there is a low probability of surviving, since
approximately 75% of all patients are diagnosed at stages III and IV of
the disease, and poor prognosis is associated with late diagnosis of the
disease at its advanced stages. Resistance to currently-available
chemotherapeutic agents is another major problem. Although complete
clinical response is achieved in 75% of patients after initial treatment,
most will develop recurrent disease and require re-treatment.
Unfortunately, the overwhelming majority will eventually develop
chemoresistance and succumb to the disease.

[0005]Chemoresistance is a complex phenomenon that involves a change in
the expression and biological activity of several genes and gene
products. The genes or gene families that are expressed differently in
responsive and non-responsive individuals can be used as molecular
markers for predicting which patients might be resistant to a particular
chemotherapeutic agent or combination thereof, as is typically used
clinically. In addition, genes that are overexpressed in chemoresistant
individuals can be targets for inhibition, which may decrease resistance
of a cancer cell to a chemotherapeutic agent or agents.

[0006]As with ovarian cancer, the survival of patients with colorectal
cancer is best when the disease is diagnosed early. If the cancer is
detected early, the 5-year survival rate for colon cancer patients is
approximately 90%; unfortunately, despite increased surveillance and
preventative measures, only 37% of cancers are found at this early stage.
When the cancer has spread regionally to involve other organs the
survival rate drops to around 64% and it is drastically lowered (8%)
after the cancer has metastasized (Cancer Facts and Figures 2002;
American Cancer Society publication).

[0007]Thus, there is a need for identifying colon and ovarian cancers
early in the course of the disease process, and a particular need for
identifying cancers that are chemoresistant. More specifically, since it
is understood in the art that the behavior of cancer cells, both
regarding their tumorigenicity and their resistance to chemotherapeutic
drugs is mediated by the expression of a not completely defined set of
particular genes, there is a need in the art to identify genes and
collections or sets of genes that serve as effective molecular markers
for chemoresistance in ovarian cancer, as well as such genes or gene sets
that provide clinically effective therapeutic targets for ovarian cancer
and colon cancer.

SUMMARY OF THE INVENTION

[0008]This invention provides methods and reagents for identifying genes
involved in, or whose expression is modulated by, or wherein said
modulated expression is associated with or responsible for resistance to
chemotherapeutic drug treatment. In particular, the invention provides
genes involved with, or whose expression is so modulated, or wherein said
modulated expression is associated with or responsible for resistance to
chemotherapeutic drug treatment, as well as patterns of modulated gene
expression of a plurality of genes wherein said patterns are
characteristic of chemotherapeutic drug resistant cells, particularly
drug-resistant ovarian cancer cells. The invention further provides
methods for identifying compounds that interact with or affect expression
or activity of one or a plurality of said genes. Also provided are said
compounds that are useful as alternatives to or in conjunction with
chemotherapeutic agents for treating ovarian cancer, particularly such
cancers that are or have become resistant to conventional
chemotherapeutic treatment. The invention further provides methods and
reagents for monitoring chemotherapeutic treatment to identify patients
whose tumors are or have become resistant to chemotherapeutic agents.

[0009]The invention provides methods for identifying compounds that
decrease chemotherapeutic drug resistance and inhibit, retard or prevent
growth of tumor cells, most preferably ovarian cancer cells, that are
resistant to a chemotherapeutic agent, the method comprising the steps
of: (a) contacting with a test compound a chemotherapeutic drug resistant
cell growing in the presence of a chemotherapeutic drug for a time or at
a concentration wherein the cell is resistant to the drug and wherein the
cell expresses at least one gene that is overexpressed in chemoresistant
ovarian cancer cells, wherein the overexpressed gene is S100A10, S100A11,
Calpain 2, SPARC, MetAP2, KLK6, ARA9, Calponin 2, RNPS1, eIF5,
eIF2Bε, HSF2, WDR1, Fused toes, NM23D, ADAR1, Grancalcin, NBR1,
SAPK/Erk1, zinc finger protein-262 MYM, HYA22, MRPL4, Vinexin β,
G-CSFR, IGFBP-7, FAST kinase, TESK2, SRB1 or KIAA0082 (as identified by
the GenBank accession numbers set forth in Table 1); (b) assaying said
cells for expression or activity of one or a plurality of said genes or
gene products in the presence and absence of the test compound; and/or
(c) comparing cell growth and/or expression or activity of at least one
of the genes or gene products in the presence and absence of the test
compound, wherein a compound is identified as a compound that inhibits
chemoresistant tumor cell growth if expression or activity of the gene or
gene product in the presence of the test compound is reduced relative to
expression of the gene in the absence of the test compound, or if cell
growth is inhibited in the presence of the compound, or both. In certain
embodiments, gene expression is detected by assaying a biological sample
using an array of, inter alia, nucleic acid (gene) probes or antibodies
specific for a plurality of gene products identified herein.

[0010]Further, the invention provides methods for identifying compounds
that decrease drug resistance and inhibit, retard or prevent growth of
tumor cells, most preferably ovarian cancer cells, that are resistant to
a chemotherapeutic agent, the method comprising the steps of: a)
contacting with a test compound a cell growing in the presence of a
chemotherapeutic drug for a time or at a concentration wherein the cell
is resistant to the drug and wherein the cell expresses a gene that is
expressed at a lower level in chemoresistant ovarian cancer cells
compared with a chemo-sensitive cell, wherein the gene is HMT1, NAIP,
eEF1ε, RAB22A, NCOR2, MT1 or MPP10 (as identified by the GenBank
accession numbers set forth in Table 1); b) assaying said cells for cell
growth and/or gene expression or gene product activity in the presence
and absence of the test compound; and c) comparing expression of the gene
or activity of the gene product in the presence and absence of the test
compound, wherein a compound is identified as a compound that inhibits
chemoresistant tumor cell growth if (i) expression of the gene or
activity of the gene product in the presence of the test compound is
increased relative to expression of the gene or activity of the gene
product in the absence of the test compound, and/or (ii) if cell growth
is inhibited in the presence of the compound, and/or (iii) if cell growth
is inhibited while expression and/or activity of the gene are increased.
In certain embodiments, gene expression is detected by assaying a
biological sample using an array of, inter alia, nucleic acid (gene)
probes or antibodies specific for a plurality of gene products identified
herein.

[0011]The invention provides methods for decreasing drug resistance, or
inhibiting, retarding or preventing growth of a tumor cell, or both,
comprising the step of contacting the tumor cell with at least one
inhibitor of a cellular gene, wherein the cellular gene is S100A10,
S100A11, Calpain 2, SPARC, MetAP2, KLK6, ARA9, Calponin 2, RNPS1, eIF5,
eIF2Bε, HSF2, WDR1, Fused toes, NM23D, ADAR1, Grancalcin, NBR1,
SAPK/Erk1, zinc finger protein-262 MYM, HYA22, MRPL4, Vinexin β,
G-CSFR, IGFBP-7, FAST kinase, TESK2, SRB1 or KIAA0082 in the presence of
a chemotherapeutic drug for a time or at a concentration wherein the cell
is resistant to the drug in the absence of the cellular gene inhibitor.
In preferred embodiments, the tumor cell is a human tumor cell, and more
preferably an ovarian cancer cell. In particular aspects, one or a
plurality of the genes identified according to the invention are
inhibited with antisense RNA or siRNA molecules specifically designed to
target one or a plurality of said genes. In alternative aspects, the gene
products of said genes are inhibited using inhibitors of these proteins.

[0012]The invention provides methods for decreasing drug resistance of a
tumor cell, comprising the step of contacting the tumor cell with at
least one compound that increases expression or activity of a cellular
gene, wherein the cellular gene is HMT1, NAIP, eEF1ε, RAB22A,
NCOR2, MT1, or MPP10, in the presence or absence of a chemotherapeutic
drug for a time or at a concentration wherein the cell is resistant to
the drug in the absence of the compound that increases expression or
activity of a HMT1, NAIP, eEF1ε, RAB22A, NCOR2, MT1, or MPP10. In
preferred embodiments, the tumor cell is a human tumor cell, and more
preferably an ovarian cancer cell. The invention also provides methods
for inhibiting, retarding or preventing growth of a tumor cell,
comprising the step of contacting the tumor cell with at least one
compound that increases expression or activity of a cellular gene,
wherein the cellular gene is HMT1, NAIP, eEF1ε, RAB22A, NCOR2,
MT1, or MPP10, in the presence or absence of a chemotherapeutic drug for
a time or at a concentration wherein cell proliferation is slowed or
inhibited in the presence of the compound that increases expression or
activity of a HMT1, NAIP, eEF1ε, RAB22A, NCOR2, MT1, or MPP10
compared with cell proliferation in the absence said compound. In
preferred embodiments, the tumor cell is a human tumor cell, and more
preferably an ovarian cancer cell.

[0013]In another aspect, the invention provides methods for inhibiting,
retarding or preventing growth of a tumor cell, most preferably an
ovarian cancer cell, the method comprising the step of contacting the
tumor cell with a combination of a chemotherapeutic agent or agents and
at least one inhibitor of a cellular gene, wherein the cellular gene is
S100A10, S100A11, Calpain 2, SPARC, MetAP2, KLK6, ARA9, Calponin 2,
RNPS1, eIF5, eIF2Bε, WDR1, Fused toes, NM23D, Grancalcin,
SAPK/Erk1, zinc finger protein-262 MYM, HYA22, Vinexin β, G-CSFR,
IGFBP-7, or KIAA0082. In a particular aspect, the cellular gene is
MetAP2, the chemotherapeutic agent is platinum-based, and the at least
one inhibitor is fumagillin or a derivative of fumagillin. In another
particular aspect, the cellular gene is Calpain 2, the chemotherapeutic
agent is platinum-based, and the at least one inhibitor is
N-acetyl-leucyl-leucyl-norleucinal (ALLN) or a derivative thereof. An
inhibitor of a cellular gene shown in Table 1 can be, for example, a
siRNA molecule or an shRNA molecule that is specifically designed to
target a gene shown in Table 1, or a small molecule inhibitor.

[0014]In another aspect, the invention provides methods for inhibiting,
retarding or preventing growth of a tumor cell, most preferably an
ovarian cancer cell, the method comprising the step of contacting the
tumor cell with a combination of a chemotherapeutic agent or agents and
at least one compound that increases expression or activity of a cellular
gene, wherein the cellular gene is HMT1, NAIP, eEF1ε, RAB22A,
NCOR2, MT1, or MPP10. In preferred embodiments, the tumor cell is a human
tumor cell, and more preferably an ovarian cancer cell.

[0015]The invention also provides methods of predicting whether an ovarian
cancer patient's tumor will be resistant to chemotherapeutic treatment,
comprising the steps of: (a) detecting an amount of one or a plurality of
expressed genes or gene products encoded thereby in a biological sample
taken from the patient, wherein the expressed gene is S100A10, S100A11,
Calpain 2, SPARC, MetAP2, KLK6, ARA9, Calponin 2, RNPS1, eIF5,
eIF2Bε, HSF2, WDR1, Fused toes, NM23D, ADAR1, Grancalcin, NBR1,
SAPK/Erk1, zinc finger protein-262 MYM, HYA22, MRPL4, Vinexin β,
G-CSFR, IGFBP-7, FAST kinase, TESK2, SRB1, or KIAA0082; (b) detecting an
amount of the one or the plurality of expressed genes or gene products
encoded thereby in a control sample comprising a nontumor tissue sample,
most preferably from the tissue of origin of the tumor, or a tissue
sample from a patient that responded well to chemotherapy, corresponding
to the one or plurality of expressed genes or gene products detected in
subpart (a), wherein the expressed gene is S100A10, S100A11, Calpain 2,
SPARC, MetAP2, KLK6, ARA9, Calponin 2, RNPS1, eIF5, eIF2Bε, HSF2,
WDR1, Fused toes, NM23D, ADAR1, Grancalcin, NBR1, SAPK/Erk1, zinc finger
protein-262 MYM, HYA22, MRPL4, Vinexin β, G-CSFR, IGFBP-7, FAST
kinase, TESK2, SRB1, or KIAA0082; and (c) comparing the amount of the
expressed gene or gene product measured in step (a) with the amount of
the expressed gene or gene product detected in step (b), wherein the
patient is predicted to be resistant to chemotherapy if the amount
detected in step (a) is greater than the amount detected in step (b) by a
factor of at least 20%. In a particular aspect, the biological sample is
a tumor sample. In a particular aspect, the control sample is a
biological sample obtained from a cancer patient who is responsive to
chemotherapy. In certain embodiments, gene expression is detected by
assaying a biological sample using an array of, inter alia, nucleic acid
(gene) probes or antibodies specific for a plurality of gene products
identified herein.

[0016]In a particular aspect, the method predicts that a patient will be
resistant to platinum-based chemotherapy when the measured amount of
MetAP2 expressed in the biological sample from the cancer patient is
greater than the amount detected in a chemotherapeutic drug responsive
individual or in ovarian tissue from an individual without ovarian
cancer.

[0017]The invention also provides methods of predicting whether an ovarian
cancer patient's tumor will be resistant to chemotherapeutic treatment,
comprising the steps of:

[0018](a) detecting an amount of one or a plurality of expressed genes or
gene products encoded thereby in a biological sample taken from the
patient, wherein the expressed gene is HMT1, NAIP, eEF1ε, RAB22A,
NCOR2, MT1, or MPP10; (b) detecting an amount of the one or the plurality
of expressed genes or gene products encoded thereby in a control sample
corresponding to the one or plurality of expressed genes or gene products
detected in subpart (a), wherein the expressed gene is HMT1, NAIP,
eEF1ε, RAB22A, NCOR2, MT1 or MPP10; and (c) comparing the amount
of the expressed gene or gene product measured in step (a) with the
amount of the expressed gene or gene product detected in step (b),
wherein the patient is predicted to be resistant to chemotherapy if the
amount detected in step (a) is less than the amount detected in step (b)
by a factor of at least 20%, more preferably at least 50%. In a
particular aspect, the control sample is a biological sample obtained
from a cancer patient who is responsive to chemotherapy. In a particular
aspect, the biological sample is a tumor sample. In certain embodiments,
gene expression is detected by assaying a biological sample using an
array of, inter alia, nucleic acid (gene) probes or antibodies specific
for a plurality of gene products identified herein.

[0019]The invention further provides methods for monitoring disease
progression in an ovarian cancer patient, particularly an ovarian cancer
patient being treated with a chemotherapeutic treatment, comprising the
steps of: (a) detecting an amount of one or a plurality of expressed
genes or gene products encoded thereby in a biological sample taken from
the patient, wherein the expressed gene is S100A10, S100A11, Calpain 2,
SPARC, MetAP2, KLK6, ARA9, Calponin 2, RNPS1, eIF5, eIF2Bε, HSF2,
WDR1, Fused toes, NM23D, ADAR1, Grancalcin, NBR1, SAPK/Erk1, zinc finger
protein-262 MYM, HYA22, MRPL4, Vinexin β, G-CSFR, IGFBP-7, FAST
kinase, TESK2, SRB1, or KIAA0082; (b) repeating step (a) using a
subsequently-collected biological sample obtained from the patient; and
(c) comparing the amount of expressed gene or gene product detected in
step (a) with the amount of expressed gene or gene product detected in
step (b), wherein disease progression is monitored by detecting changes
in the amount of expressed gene or gene products in the
subsequently-collected biological sample compared with the biological
sample taken in step (a), and whereby disease progression is detected
when the amount of the expressed gene or expressed gene product detected
in step (b) is greater than the amount of the expressed gene or gene
product detected in step (a). In certain embodiments, the patient
undergoes chemotherapeutic or other treatment during the period between
detecting the amount of gene expression in step (a) and the amount
detected in step (b). In a particular aspect, the biological sample is a
tumor sample. In preferred embodiments, gene expression is detected by
assaying a biological sample using an array of, inter alia, nucleic acid
(gene) probes or antibodies specific for a plurality of gene products
identified herein.

[0020]The invention further provides methods for monitoring disease
progression in an ovarian cancer patient, particularly an ovarian cancer
patient being treated with a chemotherapeutic treatment, comprising the
steps of: (a) detecting an amount of one or a plurality of expressed
genes or gene products encoded thereby in a biological sample taken from
the patient, wherein the expressed gene is HMT1, NAIP, eEF1ε,
RAB22A, NCOR2, MT1 or MPP10; (b) repeating step (a) using a
subsequently-collected biological sample obtained from the patient; and
(c) comparing the amount of expressed gene or gene product detected in
step (a) with the amount of expressed gene or gene product detected in
step (b), wherein disease progression is monitored by detecting changes
in the amount of expressed gene or gene products in the
subsequently-collected biological sample compared with the biological
sample taken in step (a), and whereby disease progression is detected
when the amount of the expressed gene or expressed gene product detected
in step (b) is less than or equal to the amount of the expressed gene or
gene product detected in step (a). In certain embodiments, the patient
undergoes chemotherapeutic or other treatment during the period between
detecting the amount of gene expression in step (a) and the amount
detected in step (b). In a particular aspect, the biological sample is a
tumor sample. In certain embodiments, gene expression is detected by
assaying a biological sample using an array of, inter alia, nucleic acid
(gene) probes or antibodies specific for a plurality of gene products
identified herein.

[0021]In addition, the invention provides methods for monitoring the
effectiveness of a pharmaceutical composition as an agent for treating
cancer, particularly ovarian or colon cancer in a patient comprising the
steps of: (a) detecting an amount of one or a plurality of expressed
genes or gene products encoded thereby in a biological sample taken from
a patient, wherein the expressed gene is S100A10, S100A11, Calpain 2,
SPARC, MetAP2, KLK6, ARA9, Calponin 2, RNPS1, eIF5, eIF2Bε, HSF2,
WDR1, Fused toes, NM23D, ADAR1, Grancalcin, NBR1, SAPK/Erk1, zinc finger
protein-262 MYM, HYA22, MRPL4, Vinexin β, G-CSFR, IGFBP-7, FAST
kinase, TESK2, SRB1, or KIAA0082; (b) administering an amount a
pharmaceutical composition to the patient; (c) repeating step (a) using a
subsequently-collected biological sample obtained from the patient; and
(d) comparing the amount of expressed gene or gene product detected in
step (a) with the amount of expressed gene or gene product detected in
step (c), wherein the effectiveness of the pharmaceutical composition is
monitored by detecting changes in the amount of expressed gene or gene
products in the subsequently-collected biological sample compared with
the biological sample taken in step (a), and whereby the pharmaceutical
composition is effective when the amount of the expressed gene or
expressed gene product detected in step (c) is less than the amount of
the expressed gene or gene product detected in step (a) and where growth
of the tumor is decreased (i.e., slowed, retarded or inhibited) in the
presence of the pharmaceutical composition. In a particular aspect, the
biological sample is a tumor sample. In certain embodiments, gene
expression is detected by assaying a biological sample using an array of,
inter alia, nucleic acid (gene) probes or antibodies specific for a
plurality of gene products identified herein.

[0022]The invention also provides methods for monitoring the effectiveness
of a pharmaceutical composition as an agent for treating cancer,
particularly ovarian cancer in a patient comprising the steps of: (a)
detecting an amount of one or a plurality of expressed genes or gene
products encoded thereby in a biological sample taken from a patient,
wherein the expressed gene is HMT1, NAIP, eEF1ε, RAB22A, NCOR2,
MT1 or MPP10; (b) administering an amount a pharmaceutical composition to
the patient; (c) repeating step (a) using a subsequently-collected
biological sample obtained from the patient; and (d) comparing the amount
of expressed gene or gene product detected in step (a) with the amount of
expressed gene or gene product detected in step (c), wherein the
effectiveness of the pharmaceutical composition is monitored by detecting
changes in the amount of expressed gene or gene products in the
subsequently-collected biological sample compared with the biological
sample taken in step (a), and whereby the pharmaceutical composition is
effective when the amount of the expressed gene or expressed gene product
detected in step (c) is greater than the amount of the expressed gene or
gene product detected in step (a) and whereby growth of the tumor is
decreased (i.e., slowed, retarded or inhibited) in the presence of the
pharmaceutical composition. In a particular aspect, the biological sample
is a tumor sample. In certain embodiments, gene expression is detected by
assaying a biological sample using an array of, inter alia, nucleic acid
(gene) probes or antibodies specific for a plurality of gene products
identified herein.

[0023]The invention also provides a method of detecting colon cancer
comprising the steps of: (a) obtaining a biological sample from an
animal, preferably a human; (b) detecting an amount of one or a plurality
of expressed genes or gene products encoded thereby in the biological
sample, wherein the expressed gene is S100A10, S100A11, Calpain 2, SPARC,
or MetAP2; (c) detecting an amount of the one or plurality of expressed
genes or gene products detected in a control sample comprising a nontumor
colon tissue sample; (d) comparing the amount the one or plurality of
expressed genes or gene products from step (b) with the amount in step
(c), wherein colon cancer is detected if there is a difference in the
amount in step (b) compared with the amount in step (c). The difference
detected can be overexpression of the one or plurality of said genes, or
can be lack of or underexpression of the one or plurality of said genes,
in the biological sample taken in step (a) compared with the biological
sample taken in step (c). For example, colon cancer is detected if the
amount of S100A10, S100A11, SPARC, and/or MetAP2 is greater in step (b)
compared with the amount in step (c), and/or if the amount of Calpain 2
is less in step (b) than the amount in step (c). In certain embodiments,
the animal is a human, preferably a human having colon cancer.
Preferably, the biological sample is a colon tissue sample, more
preferably a polyp and yet more preferably an adematous polyp, which are
commonly used in the art as tissue samples for colon screening
activities. In certain embodiments, gene expression is detected by
assaying a biological sample using an array of, inter alia, nucleic acid
(gene) probes or antibodies specific for a plurality of gene products
identified herein.

[0024]In yet another embodiment, the invention provides methods for
diagnosing cancer and/or chemotherapeutic drug resistance in an animal,
preferably a human, comprising the step of detecting a pattern of changes
in amount of two or a plurality of expressed genes or gene products
encoded thereby. In a particular embodiment, the expressed genes are
genes shown in Table 1. Generally, these methods of the invention
comprise the steps of: (a) obtaining a biological sample from an animal,
preferably a human; (b) detecting an amount of two or a plurality of
expressed genes or gene products encoded thereby in the biological
sample, wherein the expressed gene is shown in Table 1; (c) detecting an
amount of the two or plurality of expressed genes or gene products
detected in a control sample; (d) determining a pattern of changes in the
amount of the two or a plurality of expressed genes or gene products
encoded thereby by comparing the amount the two or plurality of expressed
genes or gene products from step (b) with the amount in step (c), wherein
the pattern is associated with a cancer, for example colon or ovarian
cancer, or drug resistance, for example resistance to cis-platin. In
certain embodiments, gene expression is detected by assaying a biological
sample using an array of, inter alia, nucleic acid (gene) probes or
antibodies specific for a plurality of gene products identified herein.

[0025]The invention also provides methods for detecting chemotherapeutic
drug resistance in an animal with ovarian cancer, the method comprising
the steps of (a) detecting an amount of a plurality of expressed genes or
gene products encoded thereby in a biological sample taken from the
animal, wherein the expressed gene is S100A10, S100A11, Calpain 2, SPARC,
MetAP2, KLK6, ARA9, Calponin 2, RNPS1, eIF5, eIF2Bε, HSF2, WDR1,
Fused toes, NM23D, ADAR1, Grancalcin, NBR1, SAPK/Erk1, zinc finger
protein-262 MYM, HYA22, MRPL4, Vinexin β, G-CSFR, IGFBP-7, FAST
kinase, TESK2, SRB1, or KIAA0082; (b) detecting an amount of the said
plurality of expressed genes or gene products encoded thereby in a
control sample comprising nontumor ovarian tissue or tumor tissue from a
patient responsive to chemotherapy, corresponding to the plurality of
expressed genes or gene products detected in subpart (a), wherein the
expressed gene is S100A10, S100A11, Calpain 2, SPARC, MetAP2, KLK6, ARA9,
Calponin 2, RNPS1, eIF5, eIF2Bε, HSF2, WDR1, Fused toes, NM23D,
ADAR1, Grancalcin, NBR1, SAPK/Erk1, zinc finger protein-262 MYM, HYA22,
MRPL4, Vinexin β, G-CSFR, IGFBP-7, FAST kinase, TESK2, SRB1, or
KIAA0082; and (c) comparing the amount of the expressed gene or gene
product measured in step (a) with the amount of the expressed gene or
gene product detected in step (b), wherein the patient is predicted to be
resistant to chemotherapy if the amount detected in step (a) is greater
than the amount detected in step (b) by a factor of at least 20%. As
provided herein, the plurality of said genes wherein the amount detected
in step (a) is greater than the amount detected in step (b) by a factor
of at least 20% defines a gene expression pattern specific for tumor
samples that are resistant to a chemotherapeutic drug. In a particular
aspect, the control sample is a biological sample obtained from a cancer
patient who is responsive to chemotherapy. Preferably, expression of one
or a plurality of said genes is greater in the tumor sample detected in
step (a) than in the control sample detected in step (b). In preferred
embodiments, the animal is a human, most preferably a human cancer
patient. As disclosed herein, the invention further provides a gene
expression pattern that predicts resistance to said chemotherapeutic drug
when said gene expression pattern is detected. In preferred embodiments,
gene expression is detected by assaying a biological sample using an
array of, inter alia, nucleic acid (gene) probes or antibodies specific
for a plurality of gene products identified herein.

[0026]Advantageously, some genes identified herein have never been
recognized as associated with either ovarian or colon cancer and may
prove to be novel targets for intervention with these diseases.

[0027]Specific preferred embodiments of the invention will become evident
from the following more detailed description of certain preferred
embodiments and the claims.

BRIEF DESCRIPTION OF THE DRAWINGS

[0028]FIG. 1 is a photograph of an autoradiogram showing the results of
Northern blot analysis, and a graphical representation of the Northern
blot results demonstrating that S100A10 is expressed at increased levels
in ovarian cancer cell lines that have increased resistance to
chemotherapeutic drug(s).

[0029]FIG. 2 is a photograph of an autoradiogram showing the results of
Northern blot analysis and a graphical representation of the Northern
blot results demonstrating that S100A11 is expressed at increased levels
in ovarian cancer cell lines that have increased resistance to
chemotherapeutic drug(s).

[0030]FIG. 3 is a photograph of an autoradiogram showing the results of
Northern blot analysis and a graphical representation of the Northern
blot results demonstrating that the mRNA levels for S100A10 and S100A11
are elevated in a patient tumor sample that is more resistant to
chemotherapy compared to a sample from a more responsive patient.

[0031]FIG. 4 is a graph representing the results of quantitative real-time
PCR demonstrating that SPARC was expressed at increased levels in
chemoresistant cell lines.

[0032]FIG. 5 is a photograph of an autoradiogram showing the results of
Northern blot analysis demonstrating that SPARC mRNA was elevated in
samples taken from a patient whose cancer had recurred.

[0033]FIG. 6 is a photograph of an autoradiogram showing the results of
Northern blot analysis and a graphical representation of the Northern
blot results demonstrating that the levels of Calpain 2 mRNA were
increased in the chemoresistant ovarian cancer cell lines.

[0034]FIG. 7 is a graphical representation of Northern blot results
demonstrating that Grancalcin mRNA levels were elevated in chemoresistant
cell lines compared to cell lines sensitive to treatment with cis-platin.

[0035]FIG. 8 is a photograph of an autoradiogram showing the results of
Western blot analysis and a graphical representation of the Western blot
results demonstrating that expression of MetAP2 protein was elevated in
the highly chemoresistant cell line OVCA 429 and down-regulated in the
Hey cell line, which is sensitive to cis-platin treatment.

[0036]FIG. 9 is a photograph of an autoradiogram showing the results of
Northern blot analysis and a graphical representation of the Northern
blot results demonstrating expression of MetAP2 mRNA in tissue samples
obtained from three patients with different levels of resistance to
cis-platin-based chemotherapy. MetAP2 is most elevated in the sample from
the patient having the most resistant tumor (CAP3) compared to patients
with intermediate (CAP2) and low (CAP1) levels of resistance to
chemotherapy.

[0037]FIG. 10 is a photograph of an autoradiogram showing the results of
Northern blot analysis and a graphical representation of the Northern
blot results demonstrating that two transcripts of eIF5 were detected and
that expression levels of both were elevated in ovarian cancer cell lines
with the highest level of resistance to cis-platin. FIG. 11 is a
photograph of an autoradiogram showing the results of Northern blot
analysis and a graphical representation of the Northern blot results
demonstrating expression of eIF5 in a chemoresistant patient tumor sample
before (CAP2) and after the recurrence of the tumor (CAP2+).

[0038]FIG. 12 is a photograph of an autoradiogram showing the results of
Northern blot analysis and a graphical representation of the Northern
blot results demonstrating that mRNA for eIF2Bε was elevated in
chemoresistant cell lines.

[0039]FIG. 13 is a photograph of an autoradiogram showing the results of
Northern blot analysis and a graphical representation of the Northern
blot results demonstrating that eEF1ε mRNA was down-regulated in
ovarian cancer cell lines that were the most resistant to cis-platin.

[0043]FIG. 17 is a photograph of an autoradiogram showing the results of
Northern blot analysis and a graphical representation of the Northern
blot results demonstrating that levels of expression of KLK6 were
elevated in tested cis-platin resistant cell lines.

[0044]FIG. 18 is a photograph of an autoradiogram showing the results of
Northern blot analysis and a graphical representation of the Northern
blot results demonstrating that the expression of HMT1 was down-regulated
in cells that are resistant to cis-platin.

[0045]FIG. 19 is a photograph of an autoradiogram showing the results of
Northern blot analysis and a graphical representation of the Northern
blot results demonstrating that mRNA from ARA9 was elevated in cell lines
resistant to cis-platin.

[0046]FIG. 20 is a photograph of an autoradiogram showing the results of
Northern blot analysis and a graphical representation of the Northern
blot results demonstrating that expression of Calponin 2 was elevated in
chemoresistant cell lines compared to chemosensitive ovarian cancer cell
lines.

[0047]FIG. 21 is a photograph of an autoradiogram showing the results of
Northern blot analysis and a graphical representation of the Northern
blot results demonstrating that neuronal apoptosis inhibitory protein
gene expression was decreased in cell lines most resistant to cis-platin.

[0048]FIG. 22 is a photograph of an autoradiogram showing the results of
Northern blot analysis and a graphical representation of the Northern
blot results demonstrating that RNPS 1 levels were elevated in cell lines
resistant to cis-platin.

[0049]FIG. 23 is a photograph of an autoradiogram showing the results of
Northern blot analysis and a graphical representation of the Northern
blot results demonstrating that mRNA levels for HSF2 were elevated in the
chemoresistant cell lines.

[0050]FIG. 24 is a photograph of an autoradiogram showing the results of
Northern blot analysis and a graphical representation of the Northern
blot results demonstrating that mRNA for WDR1 was elevated in
chemoresistant cell lines compared to chemosensitive cell lines.

[0051]FIG. 25 is a photograph of an autoradiogram showing the results of
Northern blot analysis and a graphical representation of the Northern
blot results demonstrating that levels of Ft1 mRNA was elevated in cell
lines that are resistant to cis-platin.

[0052]FIG. 26 is a photograph of an autoradiogram showing the results of
Northern blot analysis and a graphical representation of the Northern
blot results demonstrating that NME4 mRNA was elevated in the
chemoresistant cell lines.

[0053]FIG. 27 shows a graphical representation of Northern blot results
demonstrating that ADAR1 mRNA was elevated in cell lines that are
resistant to cis-platin.

[0054]FIG. 28 shows a graphical representation of Northern blot results
demonstrating that NBR1 mRNA was elevated in OVCA 429, the most
chemoresistant cell line compared to the other cell lines tested.

[0055]FIG. 29 shows a graphical representation of Northern blot results
demonstrating that mRNA for zinc finger protein 262 was elevated in the
most cis-platin resistant cell line compared to the other cell lines
tested. FIG. 30 shows a graphical representation of Northern blot results
demonstrating that MRPL4 mRNA was elevated in chemoresistant cell lines.

[0063]FIG. 38 shows a Northern blot analysis and a graphical
representation of results demonstrating that the mRNA for NCOR2 was
reduced in cis-platin resistant cell lines compared to sensitive ones.

[0064]FIG. 39 shows the rankings of five ovarian cancer cell lines
according to their level of sensitivity to cis-platin based on the
results of MTT assays.

[0065]FIG. 40 (upper panel) depicts a graph showing the effects of
increasing concentration of fumagillin on OVCA 429 cell survival after 4
hours of exposure to the drug. The bottom panel depicts a graph showing
that there was an enhancement of the effect of cis-platin in the presence
of 0.1 μg/ml fumagillin but not when the cells were treated with
cis-platin in the presence of 10 μg/ml fumagillin for 4 hours.

[0066]FIG. 41 (upper panel) depicts a graph showing the effects of
increasing concentration of fumagillin on OVCA 429 cell survival after 8
hours of exposure to the drug.

[0067]FIG. 41 (lower panel) depicts a graph showing that there was an
enhancement of the effect of cis-platin in the presence of 0.1 μg/ml
fumagillin but not when the cells were treated with cis-platin in the
presence of 10 μg/ml fumagillin for 8 hours.

[0068]FIG. 42 (upper panel) depicts a graph showing the effects of
increasing concentration of fumagillin on OVCA 429 cell survival after 24
hours of exposure to the drug.

[0069]FIG. 42 (lower panel) depicts a graph showing that there was an
enhancement of the cytotoxic effect of cis-platin in the presence of 0.1
μg/ml fumagillin but not when the cells were treated with cis-platin
in the presence of 10 μg/ml fumagillin for 24 hours. FIG. 43 is a
schematic representation of three siRNAs (#1, SEQ ID NO: 4; #2, SEQ ID
NO: 5; and #3, SEQ ID NO: 6) designed to target different regions of the
MetAP-2 messenger RNA.

[0070]FIG. 44 shows the effect of siRNA #1 on the levels of MetAP-2
expression in OVCA 429 as determined by quantitative real-time PCR.

[0071]FIG. 45 is a graph representing the quantitation of cell survival as
determined by MTT assays after exposing OVCA 429 to cis-platin in the
presence of siRNA #1.

[0072]FIG. 46 is a photograph of 96-well plates containing OVCA 429 cells
after performing the MTT assay (the quantitation is shown in FIG. 45)
that shows the effects of cis-platin on these cells transfected with
MetAP-2 siRNA #1.

[0073]FIG. 47 is a schematic representation of three siRNAs (# 1, SEQ ID
NO: 1; #2, SEQ ID NO: 2; and #3, SEQ ID NO: 3) that were designed to
target different regions of the SPARC message.

[0074]FIG. 48 is a graph representing the results of quantitative
real-time PCR analysis of SPARC expression in OVCA 429 cells transfected
with the siRNAs shown in FIG. 47.

[0075]FIG. 49 is a photograph of 96-well plates containing OVCA 429 cells
after performing the MTT assay to determine the effects of cis-platin on
OVCA 429 cells in the presence of SPARC siRNA #2.

[0076]FIG. 50 is a graph representing the effects of siRNA-mediated
reduction of SPARC gene expression on cis-platin sensitivity in OVCA 429
cells.

[0077]FIG. 51 shows a graphical representation of Northern blot results
demonstrating that MT1 mRNA was highly elevated in the cell line most
sensitive to cis-platin (Hey).

[0079]FIG. 53 is a graph representing the effects of siRNA-mediated
reduction of Calpain 2 gene expression in OVCA 429 cells.

[0080]FIG. 54 is a graph representing the effects of Calpain 2 siRNA #3 on
cis-platin sensitivity in OVCA 429 cells.

[0081]FIG. 55 is a graph representing the effects of the Calpain 2
inhibitor ALLN on cis-platin sensitivity in OVCA 429 cells.

[0082]FIG. 56 is a graph representing the effects of siRNA-mediated
reduction of SA100A10 gene expression on cis-platin sensitivity in OVCA
429 cells.

[0083]FIG. 57 is a graph representing the effects of siRNAs on S100A1l
gene mRNA expression levels in OVCA 429 cells.

[0084]FIG. 58 is a graph representing the expression levels of MetAp-2
mRNA in normal and colon cancer cell cDNA.

[0085]FIG. 59 is a graph representing the expression levels of SPARC mRNA
in normal and colon cancer cell cDNA.

[0086]FIG. 60 is a graph representing the expression levels of S100A11
mRNA in normal and colon cancer cell cDNA. FIG. 61 is a graph
representing the expression levels of S100A10 mRNA in normal and colon
cancer cell cDNA.

[0087]FIG. 62 is a graph representing the expression levels of Calpain-2
mRNA in normal and colon cancer cell cDNA.

[0088]FIG. 63 shows the volume of the tumor as a function of body weight
of two nude mice injected with OVCAR-3 cells (15 million/injection,
obtained from the AMERICAN TYPE CULTURE COLLECTION, Manassas, Va.,
Accession No. HTB-161) and treated after 35 days with cis-platin at 4
μg/kg body weight administered by IP injection 3 times a week for 2
weeks, followed by 1 week with no treatment or treated with saline
solution alone as control.

[0089]FIG. 64 is a graph that shows stable expression of siRNAs against
either Calpain 2 or S100A11 in OVCAR-3 cells. In the control lanes, the
expression of both mRNAs was measured in cells without treatment or the
irrelevant GFP siRNA. mRNA expression of Calpain 2 and S100A11 was
greatly reduced in the relevant siRNA lanes.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0090]The invention provide methods for inhibiting, retarding or
preventing growth of a tumor cell, comprising the step of contacting the
tumor cell in the presence of a chemotherapeutic drug at a concentration
to which the cell is resistant with at least one modulator of expression
or activity of one or a plurality of cellular genes, wherein the cellular
gene is a gene shown in Table 1, and wherein contacting the tumor cell
with said gene expression modulator reduces, inhibits, retards or
prevents drug resistance in the tumor cell. The tumor cell can be for
example, an ovarian cancer. In one embodiment, the tumor cell can be
contacted in vivo (e.g. a cell that has not been removed from a patient).

[0091]The term "biological sample" as used herein includes, but is not
limited to, a tissue or bodily fluid obtained from an animal, preferably
a mammal and most preferably a human. For example, a biological sample
can be biopsy material, bone marrow samples, blood, blood plasma, serum
or cellular fraction thereof, urine, saliva, tears, or cells derived from
a biological source. In one embodiment, the mammal is a human suspected
of having or previously diagnosed as having or in need of screening for a
cancer, in particular ovarian or colon cancer. In certain embodiments, a
biological sample is a tumor sample.

[0092]As used herein, the term "ovarian cancer" will be understood to
refer generally to epithelial ovarian cancer, which comprises some 80% of
all diagnosed human cancer from ovarian tissues. The remainder,
comprising germline-derived ovarian cancer and clear cell ovarian cancer,
are rare, and frequently misdiagnosed. Insofar as the changes in gene
expression disclosed herein are also found in these minor tumor types,
the methods and compositions of the inventions apply thereto.

[0093]As used herein, a "modulator" of gene expression or gene product
activity can be any chemical compound, nucleic acid molecule, peptide or
polypeptide that can cause an increase or decrease in expression of a
gene or activity of a gene product. In certain embodiments, a modulator
of the invention is a compound that causes an increase in the expression
or activity of one or a plurality of cellular genes whose expression or
activity is decreased in tumor cells that are resistant to
chemotherapeutic agents; such modulators are termed "activators" herein.
In other embodiments, a modulator is an inhibitor of expression or
activity of one or a plurality of cellular genes, particularly a gene
whose expression is increased in tumor cells that are resistant to
chemotherapeutic agents; such modulators are termed "inhibitors" herein.

[0094]As used herein, an "inhibitor" can be any chemical compound, nucleic
acid molecule, peptide or polypeptide such as an antibody against a gene
product that can reduce activity of a gene product or directly interfere
with expression of a gene. An inhibitor of the invention, for example,
can inhibit the activity of a protein that is encoded by a gene either
directly or indirectly. Direct inhibition can be accomplished, for
example, by binding to a protein and thereby preventing the protein from
binding an intended target, such as a receptor. Indirect inhibition can
be accomplished, for example, by binding to a protein's intended target,
such as a receptor or binding partner, thereby blocking or reducing
activity of the protein. Furthermore, an inhibitor of the invention can
inhibit a gene by reducing or inhibiting expression of the gene, inter
alia by interfering with gene expression (transcription, processing,
translation, post-translational modification), for example, by
interfering with the gene's mRNA and blocking translation of the gene
product or by post-translational modification of a gene product, or by
causing changes in intracellular localization.

[0095]As used herein, an "activator" can be any chemical compound, nucleic
acid molecule, peptide or polypeptide can enhance activity of a gene
product (e.g., by stabilizing the gene product, preventing its
proteolytic degradation or increasing its enzymatic or binding activity
or directly activating expression of a gene). An activator of the
invention can increase the activity of a protein that is encoded by a
gene either directly or indirectly. Direct activation can be
accomplished, for example, by binding to a protein and thereby enhancing
binding of the protein to an intended target, such as a receptor.
Indirect activation can be accomplished, for example, by binding to a
protein's intended target, such as a receptor or binding partner, and
enhancing activity, e.g. by increasing the effective concentration of the
target. Furthermore, an activator of the invention can activate a gene by
increasing expression of the gene, e.g., by increasing gene expression
(transcription, processing, translation, post-translational
modification), for example, by stabilizing the gene's mRNA or blocking
degradation of the mRNA transcript, or by post-translational modification
of a gene product, or by causing changes in intracellular localization.

[0096]As described herein, the expression of several genes in
chemoresistant ovarian tumor cells differs substantially from expression
thereof in chemosensitive ovarian tumor cells. Table 1 provides a list of
such genes identified using methods described in the Examples below. The
Table also summarizes expression patterns of these genes in cells
sensitive or resistant to cis-platin, a widely used chemotherapeutic
agent.

[0097]The chromosomal locations that appear in bold type in Table 1 have
been reported to be associated with ovarian cancer (Pejoic, 1995, Ann.
Med. 27:73-78).

[0098]In one embodiment, an inhibitor of a cellular gene shown in Table 1
can be, for example, a small molecule inhibitor, an antibody, a nucleic
acid such as an antisense nucleic acid, a short interfering RNA (siRNA)
molecule, or a short hairpin RNA (shRNA) molecule. In addition, such
inhibitors can be specifically designed using the methods described
herein or using methods known in the art. For example, antibodies,
particularly neutralizing antibodies and preferably monoclonal
antibodies, to proteins encoded by a gene shown in Table 1 can be
generated by conventional means as described, for example, in
"Antibodies: A Laboratory Manual" by Harlow and Lane (Cold Spring Harbor
Press, 1988), which is hereby incorporated by reference.

[0099]In a particular embodiment, an inhibitor of the invention is a siRNA
that binds to mRNA encoding a target gene, wherein the target gene is a
gene shown in Table 1.

[0101]RNA, but further encompasses chemically modified nucleotides and
non-nucleotides having RNAi capacity or activity.

[0102]Short interfering RNA mediated RNAi has been studied in a variety of
systems. Fire et al. were the first to observe RNAi in C. elegans (1998,
Nature 391:806). Wianny and Goetz described RNAi mediated by dsRNA in
mouse embryos (1999, Nature Cell Biol. 2:70). Hammond et al. described
RNAi in Drosophila cells transfected with dsRNA (2000, Nature 404:293).
Elbashir et al. described RNAi induced by introduction of duplexes of
synthetic 21-nucleotide RNAs in cultured mammalian cells including human
embryonic kidney and HeLa cells (2001, Nature 411:494). These studies
have shown that siRNA duplexes comprising 21 nucleotides are most active
when containing two nucleotide 3'-overhangs. Furthermore, substitution of
one or both siRNA strands with 2'-deoxy or 2'-O-methyl nucleotides
abolishes RNAi activity, whereas substitution of 3'-terminal siRNA
nucleotides with deoxynucleotides was shown to be tolerated. Mismatch
sequences in the center of the siRNA duplex were also shown to abolish
RNAi activity. In addition, these studies also indicate that the position
of the cleavage site in the target RNA is defined by the 5'-end of the
siRNA guide sequence rather than the 3'-end (Elbashir et al., 2001, EMBO
J. 20:6877). Other studies have indicated that a 5'-phosphate on the
target-complementary strand of a siRNA duplex is required for siRNA
activity and that ATP is utilized in cells to maintain the 5'-phosphate
moiety on the siRNA (Nykanen et al., 2001, Cell 107:309). However siRNA
molecules lacking a 5'-phosphate are active when introduced exogenously,
suggesting that 5'-phosphorylation of siRNA constructs may occur in vivo.
Chemically-modified siRNA can be directly injected into the blood stream
for certain applications.

[0103]In certain embodiments, the invention provides expression vectors
comprising a nucleic acid sequence encoding at least one siRNA molecule
of the invention, in a manner that allows expression of the siRNA
molecule. For example, the vector can contain sequence(s) encoding both
strands of a siRNA molecule comprising a duplex. The vector can also
contain sequence(s) encoding a single nucleic acid molecule that is
self-complementary and thus forms a siRNA molecule. Non-limiting examples
of such expression vectors are described in Paul et al., 2002, Nature
Biotechnology 19:505; Miyagishi and Taira, 2002, Nature Biotechnology
19:497; Lee et al., 2002, Nature Biotechnology 19:500; and Novina et al.,
2002, Nature Medicine, online publication Jun. 3, 2003.

[0104]In certain embodiments, siRNA molecules according to the invention
can comprise a delivery vehicle, including inter alia liposomes, for
administration to a subject, carriers and diluents and their salts, and
can be present in pharmaceutical compositions. Methods for the delivery
of nucleic acid molecules are described, for example, in Akhtar et al.,
1992, Trends Cell Bio. 2:139; Delivery Strategies for Antisense
Oligonucleotide Therapeutics, ed. Akhtar, 1995, Maurer et al., 1999, Mol.
Membr. Biol. 16:129-140; Hofland and Huang, 1999, Handb. Exp. Pharmacol.,
137:165-192; and Lee et al., 2000, ACS Symp. Ser. 752:184-192, all of
which are incorporated herein by reference. Beigelman et al., U.S. Pat.
No. 6,395,713 and Sullivan et al., PCT WO 94/02595, further describe
general methods for delivery of nucleic acid molecules. These protocols
can be utilized for the delivery of virtually any nucleic acid molecule.
Nucleic acid molecules can be administered to cells by a variety of
methods known to those of skill in the art, including, but not restricted
to, encapsulation in liposomes, by iontophoresis, or by incorporation
into other delivery vehicles, such as hydrogels, cyclodextrins,
biodegradable nanocapsules, and bioadhesive microspheres, or by
proteinaceous vectors (see, for example, O'Hare and Normand,
International PCT Publication No. WO 00/53722).

[0106]In one embodiment, the invention provides an expression vector
comprising a nucleic acid sequence encoding at least one siRNA molecule
of the invention. The expression vector can encode one or both strands of
a siRNA duplex, or a single self-complementary strand that self
hybridizes into a siRNA duplex. The nucleic acid sequences encoding the
siRNA molecules can be operably linked in a manner that allows expression
of the siRNA molecule (see for example, Paul et al., 2002, Nature
Biotechnology 19:505; Miyagishi and Taira, 2002, Nature Biotechnology
19:497; Lee et al., 2002, Nature Biotechnology 19:500; and Novina et al.,
2002, Nature Medicine, online publication June 3). The term "operably
linked" is used herein to refer to an arrangement of flanking sequences
wherein the flanking sequences so described are configured or assembled
so as to perform their usual function. Thus, a flanking sequence operably
linked to a coding sequence may be capable of effecting the replication,
transcription and/or translation of the coding sequence. For example, a
coding sequence is operably linked to a promoter when the promoter is
capable of directing transcription of that coding sequence. A flanking
sequence need not be contiguous with the coding sequence, so long as it
functions correctly. Thus, for example, intervening untranslated yet
transcribed sequences can be present between a promoter sequence and the
coding sequence and the promoter sequence can still be considered
"operably linked" to the coding sequence. In another aspect, the
invention provides an expression vector comprising: a) a transcription
initiation region (e.g., eukaryotic pol I, II or III initiation region);
b) a transcription termination region (e.g., eukaryotic pol I, II or III
termination region); and c) a nucleic acid sequence encoding at least one
of the siRNA molecules of the invention; wherein said sequence is
operably linked to said initiation region and said termination region, in
a manner that allows expression and/or delivery of the siRNA molecule.
The vector can optionally include an open reading frame (ORF) for a
protein operably linked on the 5' side or the 3'-side of the sequence
encoding the siRNA of the invention; and/or an intron (intervening
sequences).

[0108]In another embodiment, the invention provides an expression vector
comprising a nucleic acid sequence encoding at least one of the siRNA
molecules of the invention, in a manner that allows expression of that
siRNA molecule. In a particular embodiment, the expression vector
comprises: a) a transcription initiation region; b) a transcription
termination region; and c) a nucleic acid sequence encoding at least one
strand of the siRNA molecule; wherein the sequence is operably linked to
the initiation region and the termination region, in a manner that allows
expression and/or delivery of the siRNA molecule.

[0109]In another embodiment the expression vector comprises: a) a
transcription initiation region; b) a transcription termination region;
c) an open reading frame; and d) a nucleic acid sequence encoding at
least one strand of a siRNA molecule, wherein the sequence is operably
linked to the 3'-end of the open reading frame; and wherein the sequence
is operably linked to the initiation region, the open reading frame and
the termination region, in a manner that allows expression and/or
delivery of the siRNA molecule.

[0110]In yet another embodiment the expression vector comprises: a) a
transcription initiation region; b) a transcription termination region;
c) an intron; and d) a nucleic acid sequence encoding at least one siRNA
molecule; wherein the sequence is operably linked to the initiation
region, the intron and the termination region, in a manner which allows
expression and/or delivery of the nucleic acid molecule.

[0111]In another embodiment, the expression vector comprises: a) a
transcription initiation region; b) a transcription termination region;
c) an intron; d) an open reading frame; and e) a nucleic acid sequence
encoding at least one strand of a siRNA molecule, wherein the sequence is
operably linked to the 3'-end of the open reading frame; and wherein the
sequence is operably linked to the initiation region, the intron, the
open reading frame and the termination region, in a manner which allows
expression and/or delivery of the siRNA molecule.

[0112]In one embodiment, growth of a tumor cell is inhibited by contacting
the tumor cell with a siRNA that inhibits SPARC. Alternatively, the tumor
cell can be contacted with the siRNA in the presence of a
chemotherapeutic drug at a concentration to which the tumor cell is
resistant. Examples of siRNA molecules that are SPARC inhibitors include,
for example:

[0113]In another embodiment, growth of a tumor cell is inhibited by
contacting the tumor cell with a siRNA that inhibits MetAP2/p67.
Alternatively, the tumor cell can be contacted with the siRNA in the
presence of a chemotherapeutic drug at a concentration to which the tumor
cell is resistant. Examples of siRNA molecules that are MetAP2/p67
inhibitors include, for example:

[0114]In another embodiment, growth of a tumor cell is inhibited by
contacting the tumor cell with a siRNA that inhibits Calpain 2.
Alternatively, the tumor cell can be contacted with the siRNA in the
presence of a chemotherapeutic drug at a concentration to which the tumor
cell is resistant. Examples of siRNA molecules that are Calpain 2
inhibitors include, for example:

[0115]In another embodiment, growth of a tumor cell is inhibited by
contacting the tumor cell with a siRNA that inhibits S100A10.
Alternatively, the tumor cell can be contacted with the siRNA in the
presence of a chemotherapeutic drug at a concentration to which the tumor
cell is resistant. Examples of siRNA molecules that are S100A10
inhibitors include, for example,

[0116]The invention also provides methods for inhibiting, retarding or
preventing growth of a tumor cell comprising the step of contacting the
tumor cell with a combination of a chemotherapeutic agent or agents and
at least one inhibitor of a cellular gene, wherein the cellular gene is a
gene shown in Table 1. Preferably, the tumor cell is an ovarian cancer
cell. Chemotherapeutic agents are known in the art, and include, for
example, cis-platin, paclitaxel, carboplatin, etoposide, hexamethylamine,
melphalan, and anthracyclines.

[0117]In one embodiment, the inhibitor of a cellular gene shown in Table 1
can be a small molecule inhibitor. As used herein, the term "small
molecule" refers to a molecule that has a molecular weight of less then
about 1500 g/Mol. A small molecule can be, for example, small organic
molecules, peptides or peptide-like molecules. By way of example, a small
molecule inhibitor suitable in methods of the invention can be a calpain
inhibitor, such as PD 147631,
(25,35)-trans-epoxysuccinyl-L-leucy-lamido-3-methylbutane ethyl ester
(E-64-d), N-acetyl-leucyl-leucyl-norleucinal (ALLN),
N-Acetyl-Leu-Leu-Met-al (ALLM or C19H35N.sub.3O4S), or MDL
18270; or a MetAP-2 inhibitor, such as TNP-470 (also known as AGM 1470 or
C19H28ClNO6), fumagillin (C26H34O.sub.7),
cis-fumagillin(see Kwon et al., 2000, J. Antibiot. 53:799-806),
fumagalone (see Zhou et al., 2003, J. Med. Chem. 46:3452-3454), or
ovalicin (C16H24O4). See also Han et al., 2000, Bioorganic
& Medicinal Chem. Letters 10:39-43.

[0118]In one embodiment, the inhibitor of a cellular gene shown in Table 1
can be an inhibitor as defined above. Any combination of inhibitors can
be used, for example, multiple inhibitors of a particular gene shown in
Table 1, a combination of inhibitors that each inhibit one or a plurality
of specific genes, or an inhibitor that inhibits multiple genes shown in
Table 1, or any combination thereof.

[0119]In a particular embodiment, the inventive methods comprise the step
of contacting a tumor cell with a combination of an inhibitor of MetAP2
and a platinum-based chemotherapeutic agent. A chemotherapeutic agent is
"platinum-based" if a major component of the agent is or carboplatin,
optionally in combination with taxol or cyclophosphamide. An inhibitor of
MetAP2 can be, for example, fumagillin or a derivative of fumagillin, or
a MetAP2 siRNA such as without limitation SEQ ID NO: 4, SEQ ID NO: 5, or
SEQ ID NO: 6.

[0120]The invention also provides methods for predicting whether an
ovarian cancer patient's tumor is resistant to chemotherapeutic
treatment. In these embodiments, the methods comprise the steps of: (a)
detecting an amount of one or a plurality of expressed genes or gene
products encoded thereby in a biological sample taken from the patient,
wherein the expressed gene(s) is shown in Table 1; (b) detecting an
amount of the one or the plurality of expressed genes or gene products
encoded thereby in a control sample, wherein the expressed gene is a gene
shown in Table 1; and (c) comparing the amount of the expressed gene or
gene product measured in step (a) with the amount of the expressed gene
or gene product detected in step (b), wherein the patient is predicted to
be resistant to chemotherapy if the amount detected in step (a) differs
from the amount detected in step (b) by a factor of at least 20%. In one
embodiment, the amount detected can be an amount of mRNA of a gene shown
in Table 1 or an amount of protein encoded by a gene shown in Table 1. In
another embodiment, the control sample is a biological sample from a
responsive or normal subject, i.e. an individual who responds to therapy
or one without a cancer, such as ovarian cancer. In a particular aspect,
the biological sample is a tumor sample.

[0121]In one embodiment, the expressed gene in step (a) and step (b) is
one or a plurality of S100A10, S100A11, Calpain 2, SPARC, MetAP2, KLK6,
ARA9, Calponin 2, RNPS1, eIF5, eIF2Bε, HSF2, WDR1, Fused toes,
NM23D, ADAR1, Grancalcin, NBR1, SAPK/Erk1, zinc finger protein-262 MYM,
HYA22, MRPL4, Vinexin β, G-CSFR, IGFBP-7, FAST kinase, TESK2, SRB1,
or KIAA0082, and a patient's tumor is predicted to be resistant to
chemotherapeutic treatment if the amount of the expressed gene in step
(a) is at least about 20% higher than the amount of the expressed gene in
step (b).

[0122]In a particular embodiment, the expressed gene in step (a) and step
(b) is one or a plurality of Vinexin β, G-CSFR, KLK6, SPARC, HYA22,
Calpain 2, SAPK/Erk1, SRB1, ADAR1, MRPL4, eIF5, eIF2Bε, WDR1,
NM23D, zinc finger protein-262 MYM, RNPS1, S100A10, S100A11, or MetAP2,
and patient's tumor is predicted to be resistant to chemotherapeutic
treatment if the amount of the expressed gene in step (a) is at least
about 20% higher than the amount of the expressed gene in step (b).

[0123]In another embodiment, the expressed gene in step (a) and step (b)
is one or a plurality of HMT1, NAIP, eEF1ε, RAB22A, NCOR2, MPP10,
or MT1, and a patient's tumor is predicted to be resistant to
chemotherapeutic treatment if the amount of the expressed gene in step
(a) is at least about 20%, preferably 50%, lower than the amount of the
expressed gene in step (b).

[0124]In a particular embodiment, the expressed gene in step (a) and step
(b) is one or a plurality of HMT1, eEF1ε, NAIP, RAB22A or MT1,
and patient's tumor is predicted to be resistant to chemotherapeutic
treatment if the amount of the expressed gene in step (a) is at least
about 20%, preferably 50% lower than the amount of the expressed gene in
step (b).

[0125]Thus as disclosed herein the invention provides one or a plurality
of gene expression or gene product activity patterns comprising a
plurality of said genes that are differentially (i.e., at greater or
lesser amounts) expressed or wherein the protein products encoded by said
genes have differential activity in chemotherapeutic drug resistant
ovarian tumor cells than in normal (i.e., non-tumor or chemo-sensitive)
cells. Said patterns of differential gene expression or protein product
activity are used according to the methods of the invention to detect
chemotherapeutic drug-resistant cells in a biological, most preferably a
tumor, sample, and are thus useful in predicting drug resistance in a
tumor from an individual prior to a clinician initiating a fruitless
treatment course associated with significant morbidity and mortality.

[0126]It will be understood by those of ordinary skill in the art that in
the practice of the methods of the invention, patient tumor samples can
be evaluated for expression of one or a plurality of the genes identified
herein. Each of the plurality of genes identified herein is expected to
show the differential gene expression detected using the
instantly-disclosed methods in a percentage, most preferably a high
percentage, of individual tumors isolated from specific ovarian cancer
patients. It is also expected that the confidence in the results obtained
using the predictive methods of the invention will increase with
increasing numbers of said genes assayed that display the differential
gene expression disclosed herein.

[0127]In one embodiment, the methods of the invention can be used to
screen human patients in need of treatment with chemotherapy prior to
actually treating said patients with a chemotherapeutic agent. Thus, the
inventive methods can be used to screen patients to enable a care
provider to determine whether or not treatment of said patient with a
particular chemotherapeutic agent will be ineffective. A patient who is
predicted to be non-resistant to chemotherapy based on methods of the
invention is a candidate for treatment with chemotherapy and/or an
inhibitor of a gene that is shown in Table 1. A patient who is predicted
to be resistant to chemotherapy based on a method of the invention can be
a candidate, inter alia for surgery and/or a chemotherapeutic treatment
in conjunction with an inhibitor of a gene that is shown in Table 1, or
another treatment method.

[0128]In the practice of the methods of the invention, gene expression is
detected by detecting the amount of mRNA encoding any of the genes
identified herein expressed in a biological sample, for example by
hybridization assays such as Northern blots or dot blots, or by
amplification methods such as polymerase chain reaction (PCR), more
preferably coupled with reverse transcription of the mRNA to cDNA
(RT-PCR), and even more preferably using methods known in the art for
quantitative real-time RT-PCR, as described in more detail herein. Other
approaches include detecting the amount of a protein product of said gene
or genes, in non-limiting example by assaying a biological sample using
protein-specific antisera, more preferably antibodies and even more
preferably monoclonal antibodies specific for any particular gene as
identified herein. Protein expression levels can also be determined by
assaying a biological sample for an enzymatic or antigenic activity of
the protein product. The invention also provides gene or antibody arrays
for detecting expression of genes over- or under-expressed in
chemotherapeutic drug resistant tumors, particularly ovarian and colon
tumors, wherein the arrangement of the nucleic acid probes or antibodies
in the array produce a recognizable, preferably machine-readable pattern
when a tumor sample is chemotherapeutic drug-resistant, and/or a
different, recognizable pattern when the tumor sample is chemotherapeutic
drug-sensitive.

[0129]For example, according to the methods of the invention an amount of
MetAP2 that is expressed in a biological sample from a patient is
determined and compared with an amount of MetAP2 expressed in either a
person who has ovarian cancer and responded to chemotherapy or a person
who has ovarian cancer and did not respond to chemotherapy. As used
herein, a person has "responded to" chemotherapy if a chemotherapeutic
therapy had the effect of reducing tumor size or stopping tumor growth.
Moreover, the term "responsive patient" is intended to mean one who after
surgical resection is treated with chemotherapy and remains without
clinical signs of disease for at least 6 months. If the amount of MetAP2
in the patient is equal to or less than the amount of MetAP2 expressed in
a person who has ovarian cancer and who responded to chemotherapy, the
patient is predicted to be responsive to certain chemotherapeutic agents
(e.g. platinum-based compounds). If the amount of MetAP2 in the patient
is greater than the amount of MetAP2 expressed in a person who has
ovarian cancer and who responded to chemotherapy, the patient is
predicted to be resistant to chemotherapeutic agents. Likewise, if the
amount of MetAP2 in the patient is greater than the amount of MetAP2
expressed in a person who has cancer but did not respond to chemotherapy,
the patient is predicted to be resistant to chemotherapeutic agents.

[0130]As shown in Table 1 and the Examples below, increased expression of
MetAP2 in ovarian cancer is associated with increased resistance to
cis-platin, a platinum-based chemotherapeutic agent. Consequently, in one
embodiment, methods of the invention can predict that a patient's tumor
will be resistant to platinum-based chemotherapy when the measured amount
of MetAP2 expressed in the biological sample from the cancer patient is
greater than the predetermined amount detected in a responsive
individual. In another embodiment, methods of the invention can predict
that a patient's tumor will be resistant to platinum-based chemotherapy
when the measured amount of MetAP2 expressed in the biological sample
from the cancer patient is equal to the predetermined amount detected in
the responsive individual but where the expression of one or a plurality
of genes, where the genes are: S100A10, S100A11, Calpain 2, SPARC, KLK6,
ARA9, Calponin 2, RNPS1, eIF5, eIF2Bε, HSF2, WDR1, Fused toes,
NM23D, ADAR1, Grancalcin, NBR1, SAPK/Erk1, zinc finger protein-262 MYM,
HYA22, MRPL4, Vinexin β, G-CSFR, IGFBP-7, FAST kinase, TESK2, SRB1,
or KIAA0082, is increased over expression in the responsive patient,
and/or expression of one or a plurality of the HMT1, NAIP, eEF1ε,
RAB22A, NCOR2, MT1 or MPP10 genes is decreased in comparison with
expression in the responsive patient

[0132]As set forth herein, disease progression is detected when the
expressed gene(s) in steps (a) and (b) is S100A10, S100A11, Calpain 2,
SPARC, MetAP2, KLK6, ARA9, Calponin 2, RNPS1, eIF5, eIF2Bε, HSF2,
WDR1, Fused toes, NM23D, ADAR1, Grancalcin, NBR1, SAPK/Erk1, zinc finger
protein-262 MYM, HYA22, MRPL4, Vinexin β, G-CSFR, IGFBP-7, FAST
kinase, TESK2, SRB1 or KIAA0082, and the amount of the expressed gene or
gene product detected in step (b) is greater than the amount of the
expressed gene or gene product in step (a). In certain embodiments, the
patient undergoes chemotherapeutic or other treatment during the period
between detecting the amount of gene expression in step (a) and the
amount detected in step (b).

[0133]As set forth herein, disease progression is detected when the
expressed gene(s) in steps (a) and (b) is HMT1, NAIP, eEF1ε,
RAB22A, NCOR2, MT1 or MPP10, and the amount of the expressed gene or gene
product detected in step (b) is less than the amount of the expressed
gene or gene product in step (a). In certain embodiments, the patient
undergoes chemotherapeutic or other treatment during the period between
detecting the amount of gene expression in step (a) and the amount
detected in step (b). In certain embodiments, the amount detected can be
an amount of mRNA of a gene shown in Table 1 or an amount of protein
encoded by a gene shown in Table 1.

[0134]Methods according to the invention for monitoring progression of
ovarian cancer in a patient can be used, for example, to determine if a
patient is responding positively or negatively to a certain treatment
regime, such as a chemotherapeutic treatment regime.

[0135]For example, a patient is responding negatively where expression of
S100A10, S100A11, Calpain 2, SPARC, MetAP2, KLK6, ARA9, Calponin 2,
RNPS1, eIF5, eIF2Bε, HSF2, WDR1, Fused toes, NM23D, ADAR1,
Grancalcin, NBR1, SAPK/Erk1, zinc finger protein-262 MYM, HYA22, MRPL4,
Vinexin β, G-CSFR, IGFBP-7, FAST kinase, TESK2, SRB1, or KIAA0082 is
the same or greater in a biological sample taken from a patient at a time
after the patient started a certain treatment regime compared with the
amount of the expressed gene in a biological sample taken before or at
the time the treatment regime was started. In another example, a patient
is responding negatively where the expression of HMT1, NAIP,
eEF1ε, RAB22A, NCOR2, MT1 or MPP10 is the same or less in a
biological sample taken from a patient some time after the patient
started a certain treatment regime compared with the amount of the
expressed gene in a biological sample taken before or at the time the
treatment regime was started. In such cases, a care provider can
determine that the treatment regime is not effective.

[0136]Alternatively, a patient is responding positively, and no change in
treatment is needed, where the expression of S100A10, S100A11, Calpain 2,
SPARC, MetAP2, KLK6, ARA9, Calponin 2, RNPS1, eIF5, eIF2Bε, HSF2,
WDR1, Fused toes, NM23D, ADAR1, Grancalcin, NBR1, SAPK/Erk1, zinc finger
protein-262 MYM, HYA22, MRPL4, Vinexin β, G-CSFR, IGFBP-7, FAST
kinase, TESK2, SRB1, or KIAA0082, is less in a biological sample taken
from a patient some time after the patient started a certain treatment
regime compared with the amount of the expressed gene in a biological
sample taken before or at the time the treatment regime was started.
Furthermore, a patient is responding positively where the expression of
HMT1, NAIP, eEF1ε, RAB22A, NCOR2, MT1 or MPP10 is greater in a
biological sample taken from a patient some time after the patient
started a certain treatment regime compared with the amount of the
expressed gene in a biological sample taken before or at the time the
treatment regime was started.

[0137]In addition, the invention provides methods for monitoring the
effectiveness of a pharmaceutical composition as an agent for treating
cancer in a patient comprising the steps of: (a) detecting an amount of
one or a plurality of expressed genes or gene products encoded thereby in
a biological sample taken from a patient, wherein the expressed gene is
S100A10, S100A11, Calpain 2, SPARC, MetAP2, KLK6, ARA9, Calponin 2,
RNPS1, eIF5, eIF2Bε, HSF2, WDR1, Fused toes, NM23D, ADAR1,
Grancalcin, NBR1, SAPK/Erk1, zinc finger protein-262 MYM, HYA22, MRPL4,
Vinexin β, G-CSFR, IGFBP-7, FAST kinase, TESK2, SRB1, or KIAA0082;
(b) administering an amount of a pharmaceutical composition to the
patient; (c) repeating step (a) using a subsequently-collected biological
sample obtained from the patient; and (d) comparing the amount of
expressed gene or gene product detected in step (a) with the amount of
expressed gene or gene product detected in step (c), wherein the
effectiveness of the pharmaceutical composition is monitored by detecting
changes in the amount of expressed gene or gene products in the
subsequently-collected biological sample compared with the biological
sample taken in step (a). If gene expression is greater than or equal to
the biological sample collected after treatment with the pharmaceutical
composition than in the biological sample collected prior to treatment
with the pharmaceutical composition and tumor growth has not been slowed,
retarded or inhibited during treatment with the pharmaceutical
composition, the pharmaceutical composition can be considered ineffective
for treating the patient's cancer. For example, if an amount of S100A10
mRNA is higher in samples obtained after a patient has been treated with
a pharmaceutical composition, the patient is predicted to be resistant to
further treatment with that pharmaceutical composition. Thus, the
pharmaceutical composition is considered ineffective against that
patient's cancer. In a particular aspect, the biological sample is a
tumor sample.

[0138]The invention further provides methods for monitoring the
effectiveness of a pharmaceutical composition as an agent for treating
cancer in a patient comprising the steps of: (a) detecting an amount of
one or a plurality of expressed genes or gene products encoded thereby in
a biological sample taken from a patient, wherein the expressed gene is
HMT1, NAIP, eEF1ε, RAB22A, NCOR2, MT1 or MPP10; (b) administering
an amount of a pharmaceutical composition to the patient; (c) repeating
step (a) using a subsequently-collected biological sample obtained from
the patient; and (d) comparing the amount of expressed gene or gene
product detected in step (a) with the amount of expressed gene or gene
product detected in step (c), wherein the effectiveness of the
pharmaceutical composition is monitored by detecting changes in the
amount of expressed gene or gene products in the biological sample
collected after treatment with the pharmaceutical composition compared
with the biological sample taken in step (a), i.e. collected prior to
treatment with the pharmaceutical composition. If gene expression of one
or a plurality of said genes is lower than or equal to in the
subsequently-collected biological sample (i.e., collected after treatment
with the pharmaceutical composition) than in the previously-collected
biological sample (i.e., collected prior to treatment with the
pharmaceutical composition), and tumor growth has not been slowed,
retarded or inhibited during treatment with the pharmaceutical
composition, the pharmaceutical composition can be considered ineffective
for treating the patient's cancer, and the patient is predicted to be
resistant to further treatments with that pharmaceutical composition.
Thus, the pharmaceutical composition is considered ineffective against
that patient's cancer. In a particular aspect, the biological sample is a
tumor sample.

[0139]As used herein, a "pharmaceutical composition" can be any
formulation comprising a compound (e.g. a protein, peptide,
peptidomimetic, non-peptide organic molecule, an inorganic small
molecule, or nucleic acid molecule) that is used to treat or tested for
the ability to treat a cancer, such as colon or ovarian cancer.

[0140]The invention also provides methods for identifying compounds that
inhibit growth of a tumor cell, particularly a chemoresistant tumor cell,
and most particularly a chemoresistant ovarian cancer cell. In these
embodiments, the method comprises the steps of: (a) contacting a cell
that expresses one or a plurality of genes that are overexpressed in
chemoresistant ovarian cancer cells with a test compound, wherein the
gene is S100A10, S100A11, Calpain 2, SPARC, MetAP2, KLK6, ARA9, Calponin
2, RNPS1, eIF5, eIF2Bε, HSF2, WDR1, Fused toes, NM23D, ADAR1,
Grancalcin, NBR1, SAPK/Erk1, zinc finger protein-262 MYM, HYA22, MRPL4,
Vinexin β, G-CSFR, IGFBP-7, FAST kinase, TESK2, SRB1, or KIAA0082;
(b) detecting expression of the gene in the presence and absence of the
test compound; and (c) comparing expression of the gene in the presence
of the compound with expression of the gene in the absence of the test
compound, wherein a compound is identified as a compound that inhibits
chemoresistant tumor cell growth if expression of the gene in the
presence of the test compound is reduced relative to expression of the
gene in the absence of the test compound. In a particular aspect, the
biological sample is a tumor sample. In certain embodiments, the compound
can inhibit growth of the tumor cell in the presence of a
chemotherapeutic drug.

[0141]In addition, the invention provides methods of identifying a
compound that inhibits growth of a tumor cell, particularly a
chemoresistant tumor cell, and most particularly a chemoresistant ovarian
cancer cell comprising the steps of: (a) contacting with a test compound
a cell that expresses one or a plurality of genes that are expressed at a
lower than normal level in chemoresistant ovarian cancer cells, wherein
the gene is HMT1, NAIP, eEF1ε, RAB22A, NCOR2, MT1 or MPP10; (b)
detecting expression of the gene in the presence and absence of the test
compound; and (c) comparing expression of the gene in the presence and
absence of the test compound, wherein a compound is identified as a
compound that inhibits chemoresistant tumor cell growth if expression of
the gene in the presence of the test compound is increased relative to
expression of the gene in the absence of the test compound. In one
embodiment, the compound can inhibit growth of the tumor cell in the
presence of a chemotherapeutic drug.

[0142]The following examples, including the experiments conducted and
results achieved are provided for illustrative purposes only and are not
to be construed as limiting the invention.

[0145]Each cell line was treated with 5, 25, 50, 100 and 200 μM
cis-platin for 4, 8 and 24 hours. MTT assays were performed 96 hours
after cis-platin treatment on 96 well plates. Media was removed from the
cells and 200 μl of fresh MTT media was added to the cells and also to
blank wells to serve as controls. Cells were incubated under normal cell
culture conditions for 3-4 hours. Cells were then checked for formation
of Formazan crystals, an indication of metabolic activity. Media was
removed and 200 μl of 2-propanol were added to wells and control
wells. After all the crystals were dissolved evenly, the cells were
incubated for 20 minutes at room temperature in the dark. Results were
read on a microplate reader at 570 nm.

[0146]FIG. 39 (upper panel) shows the MTT assay results for 5 ovarian
cancer cell lines used in these studies after 4 hours of exposure to
cis-platin at various concentrations. After taking into account the
performance of each cell line over the entire range of cis-platin
concentrations and treatment times used, the cells were ranked in
decreasing level of resistance as OVCA 429<OVCA 433<HEY A8<OVCA
432<HEY (lower panel).

[0147]MTT assays involving the exposure of cells to either a siRNA or drug
inhibitor for a particular gene were conducted in essentially as
described above. The cells were pre-treated with siRNAs for 48 hours
prior to treatment with 0, 3.12, 6.25, 12.5, 25, 50, 100 and 200 μM
cis-platin for 24 hours. For combination drug treatment experiments
(fumagillin or ALLN) the cells were exposed to a combination treatment of
increasing concentrations of the drug being tested and 0, 3.12, 6.25,
12.5, 25, 50, 100 and 200 μM cis-platin for 24 hours.

[0149]The cell lines characterized in Example 1 were used to perform
micro-array analysis. Cell pellets were collected from each cell line and
RNA was isolated from the cells by dissolving the pellets in 1 ml of
TRI-Reagent (obtained from Molecular Research Center, Inc, Cincinnati,
Ohio) or Trizol (Invitrogen). The samples were then allowed to sit for 5
minutes. Phase separation was accomplished by adding 100 μl of
1-bromo-3-chloropropane (BCP) to the sample. After shaking for 15
seconds, samples were incubated at room temperature for 15 minutes and
then centrifuged for 16 minutes at 13,000 RCF at 4-25° C. The
supernatant was removed by pipette and placed into a new microfuge tube.
RNA was then precipitated by mixing the supernatant with 500 μl of
fresh isopropanol, incubated at room temperature for 10 minutes, and
centrifuged for 9 minutes at 13,000 RCF at 4-25° C. The
supernatant was then removed from the tube and the pellet was washed by
adding 1 ml of 75% ethanol to the tube, vortexing, and then centrifuging
for 6 minutes at 13,000 RCF at 4-25° C. The liquid was removed and
the pellet was air-dried for about 8 minutes. The pellet was then
dissolved in RNase-free water and placed on ice for immediate use or
stored at -80° C.

[0150]Micro-arrays (obtained from Research Genetics Inc.) containing over
5000 sequence-verified cDNA clones were used to interrogate gene
expression in these cells; all micro-array assays were conducted
according to the manufacturer's instructions. Each clone was known to be
expressed in ovarian tissue. Gene expression in the most resistant cell
line (OVCA 429) was used as a standard to which gene expression in the
other cell lines was compared. Analysis of the data revealed that OVCA
429 expressed 196 genes at increased levels and 83 genes expressed at
decreased levels compared to the more sensitive cell lines.

[0151]Genes were selected for further analysis only if they satisfied the
following criteria: an at least 2-fold difference in expression compared
to the standard as detected on duplicate membranes; differential
expression detected in 3 out of 4 cell lines compared to the standard
(OVCA 429); and expression levels consistent with each cell line's
sensitivity to cis-platin. Overexpressed genes were most highly expressed
in OVCA 429 cells and expression gradually tapered off until the lowest
level of expression was reached in the least resistant cell line HEY (and
vice versa for genes expressed at lower levels in OVCA 429 cells).

[0152]Northern blot analysis and quantitative real-time PCR analysis of
the genes that were differentially expressed (higher or lower levels) in
the most resistant cell line (OVCA 429) when compared to the other cell
lines were used to validate the microarray data and identify genes of
interest for further analysis. The identified genes are listed in Table
2, which shows the gene name, a summary of the expression pattern in the
cell lines, and the Figure that presents the results of the expression
analyses.

[0153]In order to confirm expression patterns identified by microarray
analysis, Northern blot analysis was performed using the NORTHERNMAX
Protocol (Ambion Corp., Austin, Tex.) and DNA probes were labeled using
STRIP-EZ DNA labeling kits (Ambion) according to the manufacturer's
instructions.

Quantitative Real-Time PCR

[0154]cDNA was synthesized by mixing together 1 μg total cellular RNA
isolated from ovarian cancer cell lines, 1 μl oligo dT, and water to a
final volume of 12 μl, incubating this mixture at 70° C. for
ten minutes, and then adding to the mixture 5 μl 2× Reaction
Mix, 2 μl DTT, and 1 μl of SUPERSCRIPT II Enzyme (Invitrogen). The
reaction mixture was then incubated at 42° C. for 60 minutes. cDNA
dilutions from 1:4 to 1:256 were prepared. Master mixes were prepared
with a final volume of 50 μl/well using the Qiagen QUANTITECT SYBR
Green PCR Handbook (Qiagen Corp., Valencia Calif.). For every well of a
plate that was used, 25 μl 2× QUANTITECT SYBR Green PCR Master
Mix (Qiagen), 0.3 μM of forward primer, 0.3 μM of reverse primer,
and RNase free water were added to a final volume of 45 μl.

[0156]Sequence detection was determined using the ABI Prism 7700 (Applied
Biosystems, Inc., Foster City, Calif.) sequence detection system or the
MJ Research (Waltham, Mass.) Opticon II system as follows: PCR initial
activation step was carried out for 15 minutes at 95° C.; samples
were denatured for 15 seconds at 94° C., annealed for 30 seconds
at 53° C. (55° C. when the Opticon II system was used), and
extended for 30 seconds at 72° C. (data was acquired during this
step); the PCR reaction was repeated for 50 cycles. A melting curve
analysis was prepared by adding on the following steps: 15 seconds at
95° C., 20 seconds at 60° C., and 20 seconds at 95°
C.

[0157]In addition, RNA was prepared from tissue samples obtained from
chemosensitive (i.e. responsive) and chemoresistant (i.e. non-responsive)
ovarian cancer patients who had been treated with platinum-based
chemotherapeutic agents. RNA was isolated by homogenizing 50-100 mg
tissue samples in 1 ml TRI-Reagent or Trizol until the tissues were
liquidized. The samples were then allowed to sit for 5 minutes. Phase
separation was accomplished by adding 100 μl of BCP to the sample.
After shaking for 15 seconds, samples were incubated at room temperature
for 15 minutes and then centrifuged for 9 minutes at 13,000×g
(relative centrifugal force, RCF) at 4-25° C. The supernatant was
removed by pipette and placed into a new microfuge tube. RNA was then
precipitated by mixing the supernatant with 500 μl of fresh
isopropanol, incubated at room temperature for 20 minutes, and
centrifuged for 9 minutes at 13,000 RCF at 4-25° C. The
supernatant was then removed from the tube and the pellet was washed by
adding 1 ml of 75% ethanol to the tube, vortexing, and then centrifuging
for 6 minutes at 13,000 RCF at 4-25° C. The liquid was removed and
the pellet was air-dried for about 8 minutes. The pellet was then
dissolved in RNase-free water and placed on ice for immediate use or
stored at -80° C.

[0158]Quantitative Real-Time PCR using primers for the genes shown in
Table 3 was performed to detect changes in gene expression between the
chemosensitive and chemoresistant patients. Expression of 18S RNA was
used to correct the values of the expressed genes. The results are shown
in Table 3 below. The results confirm the observations from the
experiments conducted with RNA from cell lines.

Summary of Genes Up- or Down-Regulated in Ovarian Cancer Cells that are
Resistant to Cis-Platin

Genes Encoding EF-Hand Proteins:

[0161]Five genes encoding calcium-activated EF-Hand proteins were
identified, namely, S100A10, S100A11, SPARC, Calpain 2 and Grancalcin).
Two of the four genes, S100A10 and S100A11 are located adjacent to each
other on chromosome 1 at 1q21 (Pejovic, 1995, Ann. Med. 27:73-78;
Ridinger et al., 1998, Biochimica et Biophysica Acta 1448:254-263). This
region of chromosome 1 has been reported as one of the hotbeds for
chromosomal rearrangements in ovarian cancer (Pejovic, 1995, Ann. Med.
27:73-78). The exact biological functions of S100A10 and S100A11 are
unknown. S100A10 and S100A11 are both expressed at higher levels in more
resistant ovarian cancer cell lines (see FIG. 1 and FIG. 2,
respectively). FIG. 3 shows that the mRNAs for S100A10 and S100A11 are
also elevated in a patient that is more resistant to chemotherapy
compared to a more responsive patient.

[0162]SPARC (also known as Osteonectin and BM40) is a secreted protein
(Lane and Sage, 1994, FASEB J. 8:163-173). SPARC has been shown to be
highly expressed in the stroma of neoplastic ovaries (Paley et al., 2000,
Gynecologic Oncology 78:336-341) and has been shown to induce apoptosis
in ovarian cancer cells (Yiu et al., 2001, Am. J. Pathol. 159:609-622).
However, high levels of SPARC have been detected in melanoma (Ledda et
al., 1997, J. Invest. Dermatol. 108:210-214) and colorectal cancer (Porte
et al., 1995, Int. J. Cancer 64:70-5), and also have been reported to
promote cell migration and invasion in prostate cancer (Thomas et al.,
2000, Clin. Cancer Res. 6:1140-9) and glioblastoma (Golembieski et al.,
1999, Int. J. Dev. Neurosci. 17:463-72). SPARC overexpression also
contributes to increased motility and invasion of breast cancer cells
(Briggs et al., 2002, Oncogene 21:7077-91). As shown herein, SPARC was
found to be expressed at higher levels in the more chemoresistant ovarian
cancer cell lines (FIG. 4). SPARC mRNA was also elevated in samples taken
from a patient whose tumor had recurred as shown in FIG. 5.

[0164]Grancalcin is a recently-described Ca2+-binding protein that
belongs to the penta-EF-Hand subfamily of EF-Hand proteins and
translocates to membranes upon Ca2+ binding (Lollike et al., 2001,
J. Biol. Chem. 276:17762-9). Grancalcin mRNA was found to be elevated in
cell lines more resistant to cis-platin compared to cell lines more
responsive to treatment with cis-platin (FIG. 7).

[0165]MetAP2: The expression of Methionine aminopeptidase 2 (also known as
eIF-2 associated p67) has never been linked to ovarian cancer. The
protein encoded by this gene seems to have two functions. It removes the
first methionine from newly synthesized proteins (Li and Chang, 1996,
Biochem. Biophys. Res. Commun. 227:152-9) and it also associates with
eukaryotic initiation factor 2α (eIF-2α; a GTP binding
protein) and inhibits its phosphorylation (Wu et al., 1993, J. Biol.
Chem. 268:10796-10801). Using an antibody against the MetAP2, it appears
that MetAP2 expression is elevated in the most resistant cell line OVCA
429 and down-regulated in Hey (the cell line most sensitive to
cis-platin; see FIG. 8). Furthermore, when MetAP2 mRNA expression was
examined in tissue samples obtained from three patients with different
levels of resistance to cis-platin-based chemotherapy, MetAP2 appeared to
be most elevated in the sample from the most resistant patient (CAP3 in
FIG. 9) compared to patients with intermediate (CAP2; FIG. 9) and low
(CAP1; FIG. 9) levels of resistance to chemotherapy. A drug, TNP-470,
that specifically targets MetAP2 is currently in clinical trials as an
angiogenesis inhibitor in several human tumors (Kruger and Figg, 2000,
Expert Opin. Investig. Drugs 9:1383-96). Furthermore, lowering the
cellular levels of MetAP2 using antisense oligonucleotides has been shown
to induce apoptosis (Datta and Datta, 1999, Exp. Cell Res. 246:376-83).
These observations suggest that this protein could be an important target
for therapy in ovarian cancer.

[0166]eIF5 is another central protein for translation initiation and
protein synthesis that functions as a GTPase-activator protein (Paulin et
al., 2001, Current Biol. 11:55-9; Das et al., 2001, J. Bio. Chem.
276:6720-6). Two transcripts were detected and the levels of expression
of both were elevated in ovarian cancer cell lines with the highest level
of resistance to cis-platin (FIG. 10) and in a more resistant patient
(FIG. 11).

[0169]SAPK/Erk Kinase 1 is a dual-specificity kinase that activates JNK1,
JNK2 and p38 but not Erk1 or Erk2 (Cuenda, 2000, Int. J. Biochem. Cell
Biol. 32:581-7). This gene and its protein have not heretofore been
associated with ovarian cancer. mRNA levels for this gene were found to
be elevated in more resistant cell lines compared to more sensitive cell
lines (FIG. 14).

[0170]TESK2: This serine/threonine kinase is located predominantly in the
cell nucleus. When inactive, however, it translocates to the cytoplasm.
TESK2 specifically phosphorylates cofilin (at Ser-3), a protein that,
along with actin-depolymerizing factor plays an essential role in the
rapid turnover of actin filaments and actin-based reorganization by
stimulating depolymerization and severance of actin filaments (Toshima,
2001, J. Biol. Chem. 276:31449-58). No previous link to ovarian cancer
has been reported. TESK2 mRNA is elevated in more resistant cell lines
(FIG. 15).

[0171]FAST kinase: This is a Fas-activated serine/threonine kinase, which
is thought to be involved in apoptosis mediated by Fas (Tian et al.,
1995, J. Exp. Med. 182:865-74). FAST kinase mRNA is elevated in more
chemoresistant cell lines (FIG. 16).

Others:

[0172]KLK6: This is a serine protease also known as Zyme and Neurosin.
This gene belongs to the human kallilrein gene family, which also
includes better-known molecules such as prostate specific antigen (PSA)
already being used as a marker for prostate cancer and is also being
investigated as a marker for ovarian cancer (Diamandis, 2000, Clinical
Biochem. 33:579-83). Elevated serum levels of KLK6 have been reported in
patients with ovarian cancer compared to normal controls (Diamandis,
2000, Clinical Biochem. 33:579-83). Expression levels of this gene were
elevated in the more chemoresistant cell lines tested (FIG. 17). It is
also worthwhile noting that this gene is located in another area of
frequent chromosomal re-arrangements in ovarian cancer (Pejovic, 1995,
Ann. Med. 27:73-78).

[0173]HMT1 (also known as PRMT1): This gene encodes a protein arginine
N-methyltransferase, the expression of two variants of which was found to
be down-regulated in breast cancer (Scorlis et al., 2000, Biochem.
Biophys. Res. Commun. 278:349-59). HMT1 expression was down-regulated in
cells that are more resistant to cis-platin (see FIG. 18). It is also
interesting to note that HMT1 is located on chromosome 19 at 19q13.3 in
the same chromosomal region where the gene encoding KLK6 resides.

[0174]ARA9 (also known as Aryl hydrocarbon receptor-interacting protein
(AIP) and

[0175]XAP2) is thought to play a role in AHR-mediated signaling
(Kazlauskas et al., 2002, J. Biol. Chem. 277:11795-801). mRNA from this
gene was elevated in cell lines more resistant to cis-platin (FIG. 19).
It is also located on chromosome 11 (at 11q13.3), another area with
increased frequency of chromosomal rearrangements associated with ovarian
cancer (Pejovic, 1995, Ann. Med. 27:73-78).

[0176]Calponin 2 has been studied in myoepithelial carcinomas (Mosunjac et
al., 2000, Diagn. Cytophathol. 23:151-5) but not in ovarian cancer. The
expression of Calponin 2 was slightly elevated in the more cis-platin
resistant cell lines compared to the more sensitive ovarian cancer cell
lines (FIG. 20).

[0177]Neuronal apoptosis inhibitory protein (NAIP) was found to be
slightly down-regulated in cell lines most resistant to cis-platin (FIG.
21). NAIP has never been linked to ovarian cancer (Tamm et al., 2000,
Clin. Cancer Res. 6:1796-1803).

[0178]RNA binding protein S1 (RNPS1) is a general activator of pre-mRNA
splicing and may form a complex with ASAP that is involved in promoting
apoptosis and the SART3 tumor rejection antigen (Schwerk et al., 2003,
Mol. Cell Biol. 23:2981-90; Harada et al., 2001, Int. J. Cancer
93:623-8). Its levels were found to be elevated in cell lines resistant
to cis-platin (FIG. 22).

[0180]WDR1: The WD-repeat proteins are found in all eukaryotes and play an
important role in the regulation of a wide variety of cellular functions
including signal transduction, transcription and proliferation (Li et
al., 2000, Biochem. Biophys. Res. Commun. 274:117-23). However, the exact
function of WDR1 is unknown. The mRNA for this gene was elevated in more
resistant cell lines compared to the more sensitive cell lines (FIG. 24).

[0181]Ft1: The open reading frame of this gene exhibits similarities to
ubiquitin-conjugating enzymes and in mice it maps close to the Rb-related
p130 gene (Lesche et al., 1997, Mamm. Genome 8:879-83). Cytogenetically,
Ft1 maps to chromosome 16 at region 16q12.2, an area repeatedly altered
in human cancer. Loss of heterozygosity has been reported in this
chromosomal region in ovarian cancer. The levels of Ft1 mRNA were
elevated in cell lines that are more resistant to cis-platin (FIG. 25).

[0182]NME4 (also known as nm23-h4) is a nucleoside diphosphate kinase that
is moderately over-expressed in renal cell carcinoma and strongly
over-expressed in colorectal carcinomas (Haver et al., 2001, Anticancer
Res. 21:2821-5). NME4 mRNA was elevated in the more resistant cell lines
(FIG. 26).

[0183]ADAR1: The adenosine-to-inosine RNA editing by adenosine deaminases
including ADAR1 results in the creation of alternative splicing sites or
alterations of codons and, thus, leads to functional changes in proteins
(Wang et al., 2000, Science 290:1765). It is also interesting to note
that the ADAR1 gene is located on chromosome 1 at 1q21.1-q21.2, in the
same frequently rearranged chromosomal region as S100A10 and S100A11
(Pejovic, 1995, Ann. Med. 27:73-78). ADAR1 mRNA was elevated in cell
lines that are more resistant to cis-platin (FIG. 27).

[0184]NBR1: The exact molecular function of NBR1 is unknown. (Those with
skill in the art will recognize that the usefulness of a gene in the
methods of this invention is not dependent on a detailed or exact notion
of the functional properties of a gene.) Mapping studies have revealed
that the NBR1 gene lies head-to-head with the BRCA1 gene (Whitehouse et
al., 2002, Eur. J. Biochem. 269:538-45). NBR1 has no reported association
with ovarian cancer. NBR1 mRNA was elevated in the most cis-platin
resistant cell line OVCA 429 (FIG. 28).

[0185]Zinc finger protein 262/MYM: A member of a family of genes encoding
proteins containing MYM zinc binding motif (Smedley et al., 1999,
Genomics 60:244-7). This protein has never been associated with ovarian
cancer; however, the mRNA for this gene was elevated in the most
chemoresistant cell line compared to the other cell lines tested (FIG.
29).

[0186]MRPL4: This gene and its protein have never been associated with
ovarian cancer. However, the gene is located on chromosome 19 at 19p13.2,
a region frequently rearranged in ovarian cancer (Pejovic, 1995, Ann.
Med. 27:73-78). MRPL4 mRNA was elevated in chemoresistant cell lines
(FIG. 30).

[0187]HYA22: This gene and its protein have never been associated with
ovarian cancer. However, the gene is located on chromosome 3 at 3p21.3, a
region associated with chromosomal rearrangements in ovarian cancer
(Pejovic, 1995, Ann. Med. 27:73-78; Protopopov et al., 2003, Cancer Res.
63:404-12; Senchenko et al., 2003, Oncogene 22:2984-92). HYA22 mRNA was
elevated in cell lines more resistant to cis-platin compared to the more
sensitive cell lines (FIG. 31).

[0188]Vinexin β: Also known as SCAM-1, this gene encodes an adapter
protein belonging to a family of proteins also including Vinexin β,
CAP/Ponsin and ArgBP2 (Kioka et al., 2002, Cell Structure and Function
27:1-7). Vinexin was not known in the prior art to be linked to ovarian
cancer. Vinexin β mRNA was elevated in chemoresistant cell lines
(FIG. 32).

[0189]G-CSFR: Granulocyte colony-stimulating factor receptor (G-CSFR) is
almost universally expressed in primary ovarian cancer. The expression of
its ligand, however,

[0190]G-CSF was found in the same cells in only half of the cases studied,
suggesting the presence of an autocrine system (Savarese et al., 2001,
Cancer Letters 162:105-15). In another third of the cases studied G-CSF
was found exclusively in the stroma, suggesting a paracrine system may be
present, in which mesenchymal cells may provide the ligand to cancerous
cells expressing the receptor (Savarese et al., 2001, Cancer Letters
162:105-15). A preliminary, retrospective evaluation suggested that
overall survival is worse in patients expressing the paracrine loop alone
as compared to patients whose ovarian cancer expressed an autocrine axis
(Savarese et al., 2001, Cancer Letters 162:105-15). G-CSF and its
receptor can also be co-expressed in normal ovaries and some benign
ovarian neoplasms. G-CSFR mRNA was elevated in chemoresistant cell lines
(FIG. 33).

[0191]SRB1: Also known as CLA-1, this gene encodes a receptor that
recognizes both negatively charged liposomes and apoptotic cells. Tumor
cells have been reported to participate in the uptake and removal of
apoptotic cells and bodies (Fukasawa et al., 1996, Exp. Cell Res.
222:246-50). The biological significance of these observations is poorly
understood. There has been no prior disclosure of links between the
expression of SRB1 and ovarian cancer. SRB1 mRNA was elevated in
chemoresistant cell lines (FIG. 34).

[0193]RAB22A belongs to the RAB subfamily of Ras proteins (Kauppi et al.,
2002, J. Cell Science 115:899-911). RAB22A mRNA was decreased in
chemoresistant cell lines and was elevated in cell lines more responsive
to cis-platin (FIG. 36). KIAA0082: KIAA0082 is a full-length gene for
which there is no published information. mRNA expression for this gene
was elevated in cell lines resistant to cisplatin (FIG. 37).

[0194]NCOR2: This is a co-repressor protein closely related to SMRT with a
specific interaction domain for the thyroid hormone receptor (Jepsen and
Rosenfeld, 2002, J. Cell Science 115:689-98). mRNA expression for this
gene was reduced in chemoresistant cell lines compared to cell lines
sensitive to cis-platin (FIG. 38).

[0195]MT1: The precise physiological role of Metallothionein 1L (MT1) is
unknown, however, previous studies have reported that MT levels are
elevated in cis-platin resistant human ovarian carcinoma cells (Andrews
and Howell, 1990, Cancer Cells 2:35-43) and cells transfected with the MT
gene became resistant to cis-platin (Nakano et al., 2003, Anticancer Res.
23:299-304). MTs are thought to function in sequestering cis-platin in
the cytoplasm, therefore increasing the cells ability to resist the drug
(Nakano et al., 2003, Anticancer Res. 23:299-304). Paradoxically, the
level of MT1 mRNA appeared to be highly elevated in cells most sensitive
to cis-platin (Hey) (FIG. 51).

[0196]MPP10: M-phase phosphoprotein (MPP10) is a mostly cytoplasmic
protein but can be secreted and is a component of the human U3 small
nucleolar ribonucleoprotein. The majority of this protein co-localizes
with fibrillarin (Baserga et al., 1997, Nucleic Acids Symp. Ser.
36:64-7), and is involved in rRNA processing. No association has been
reported for this gene or its product with ovarian cancer. MPP10
expression levels increased with increased sensitivity to cis-platin
(FIG. 52).

Example 3

[0197]In vitro Testing of SPARC as Therapeutic Target

[0198]SPARC was expressed at the highest levels in the most cis-platin
resistant cell line (OVCA 429) compared to the other cell lines tested
(FIG. 4). This protein is known in the art to be a calcium-binding
glycoprotein that modulates adhesion and can play an important role in
tissue remodeling and angiogenesis promoting tumor progression and
invasiveness (Ledda et al., 1997, J. Invest. Dermatol. 108:210-214).

[0199]SPARC expression was tested in human Ascites fluid samples obtained
from an individual before cyto-reductive surgery and then 9 months
following surgery when the patient's tumor had recurred (FIG. 5; the two
transcripts observed for SPARC arise due to differential polyadenylation;
Ledda et al., 1997, J. Invest. Dermatol. 108:210-214). SPARC expression
levels after surgery were greatly increased and correlated with a poor
outcome for this patient. This observation was also consistent with
findings made by other groups studying other forms of solid cancers
(Golembieski et al., 1999, Int. J. Dev. Neurosci. 17:463-72; Briggs et
al., 2002, Oncogene 21:7077-91; Huang and Wang, 2001, TRENDS in Molecular
Medicine 7:355; Lollike et al., 2001, J. Biol. Chem. 276:17762-9),
indicating that increased SPARC expression can be predictive of
chemotherapeutic treatment success and disease progression in other types
of solid cancer in addition to ovarian cancer.

[0200]In order to test whether lowering SPARC protein expression levels in
OVCA 429, the most resistant ovarian cancer cell line, would reduce its
ability to resist cis-platin, three siRNAs were designed directed against
different regions of the SPARC message. The SPARC siRNAs used were:

[0201]The siRNA experiments described herein were conducted using the
siPORT Lipid protocol (Ambion). Cells were plated 24 hours before
transfection in MEM∝ media containing 10% FBS, so that cells were
30-70% confluent at the time the experiment was performed. The siPORT and
siRNA complexes were prepared using media without FBS or antibiotics. For
siPORT, 4 microliters per well for six well plates and 0.5 microliter per
well for 96 well plates were added to media, mixed by vortexing, and
incubated at room temperature for 25 minutes. For siRNA, 1-25 nM (12.5 nM
is normally used) concentration of siRNA was used, diluted in media. The
siPORT was added to the siRNA mixture, mixed gently, and incubated at
room temperature for 20 minutes. After the cells were washed with serum
free media, cells were added to the plates (where 96 well plates were
used, cells were plated at a density of 4.5×104; where 6 well
plates were used, cells were plated at a density of 1-5×105).
The siPORT/siRNA complex was added to each plate/well and the plates were
rocked gently to distribute complex over cell surface. After incubating
for 4 hours under normal cell culture conditions, additional media
containing 10% FBS was added to the cells. After 48 hours, total RNA was
extracted.

[0202]Cells were transfected with the siRNA constructs following
instruction from the supplier (Ambion). For 6-well plates, 12.5 nM
siRNA/well was used for transfection. The OD260 readings of siRNA
were performed in duplicate at a 1:100 dilution. The readings were
averaged, then multiplied by the dilution factor and then by multiplied
by 40 (the OD260 of 1 is equal to 40 μg/ml of RNA) to get the
final concentration of siRNA in μug/ ml. The number was divided by 14
(the number of μg of RNA in 1 nmole of an average 21mer dsRNA) to get
final siRNA concentration in μM, and then converted so that the
concentration was presented in nM.

[0203]The results of these studies are shown in FIG. 48. All three siRNAs
decreased the level of SPARC mRNA in these cells. Cells treated with
siPORT only or with the sense strand of siRNA #2 alone did not show any
significant reduction in SPARC mRNA expression. A combination treatment,
which included all three siRNAs together, did have some effect, however
(FIG. 48).

[0204]The ability of OVCA 429 cells to resist cis-platin in the presence
of the SPARC siRNAs was also investigated. The cells were treated with
siPORT alone or transfected with either the sense strand of siRNA #2
alone or the complete siRNA #2. The cells were allowed to recover for 48
hours after transfection and then treated with increasing concentrations
of cis-platin or the corresponding concentrations of DMSO as a control.
The cells were exposed to the drug for 24 hours after which the drug was
removed and the cells were allowed to recover for an additional 72 hours.
The effect of this treatment on cell viability was then assessed by an
MTT assay. The results are shown in FIG. 49; FIG. 50 shows these results
after quantitation of the effects on the cells. The data suggested that
treating the cells with the complete siRNA #2 prior to exposure to
cis-platin reduced their resistance level by half (IC50˜25-50
μM for controls treated with siPORT only or sense strand only to an
IC50 of ˜12.5 μM after treating with siRNA #2).

[0205]The results of these experiments indicated that SPARC is a
therapeutic target and marker for ovarian cancer.

Example 4

[0206]In vitro Testing of MetAP2/p67 as Therapeutic Target

[0207]MetAP-2/p67 is a bifunctional protein with both functions being
important for cell growth (Li and Chang, 1996, Biochem. Biophys. Res.
Commun. 227:152-9; Wu et al., 1993, J. Biol. Chem. 268:10796-10801). In
one role, the protein binds to eukaryotic initiation factor 2 (eIF-2) and
inhibits its phosphorylation, and in the other role its C-terminus domain
has enzymatic activity catalyzing hydrolysis of N-terminal methionines
from a number of cellular proteins (Wu et al., 1993, J. Biol. Chem.
268:10796-10801). Phosphorylation of eIF-2 alters its translational
repertoire allowing different messages to be translated at different
phosphorylation states. Additionally, the methionine aminopeptidase
activity is important generally for protein function and failure to
remove N-terminal methionines often produces inactive proteins (Li and
Chang, 1996, Biochem. Biophys. Res. Commun. 227:152-9).

[0208]Fumagillin, from Aspergillus fumigatus, and its synthetic analogue
TNP-470, covalently binds to and inhibits the methionine aminopeptidase
activity of MetAP-2 but not that of the closely related MetAP-1 (Griffith
et al., 2998, Proc. Natl. Acad. Sci. USA 95:15183-8). It is also
important to note that treatment of several different cell types with
fumagillin resulted in increased expression of MetAP-2 (Wang et al.,
2000, J. Cell. Biochem. 77:465-73); it is thought that the cell adapts to
a loss of function of MetAP-2 by increasing its level of expression.

[0209]The experiments described herein suggested that OVCA 429 expressed
approximately 7 times more MetAP-2 than the cell line most sensitive to
cis-platin, Hey (FIG. 8). Northern blot analysis also confirmed that the
levels of MetAP-2 expression are also elevated in patients that are
clinically more resistant to cis-platin-based chemotherapy. FIG. 9 shows
that the level of MetAP-2 mRNA was approximately 3 fold higher in the
most resistant patient (CAP3) than in the least resistant patient (CAP1).
A patient with an intermediate level of resistance to cis-platin-based
chemotherapy also exhibited an intermediate level of MetAP-2 mRNA.

[0210]MTT-based assays were conducted in which OVCA 429 cells were treated
with fumagillin alone, cis-platin alone and with combinations of
different concentrations of cis-platin and fumagillin for 4, 8 and 24
hours. The results are shown in FIGS. 40, 41 and 42. FIG. 40 (top panel)
shows the effects of increasing concentration of fumagillin on OVCA 429
cell survival. Some cell death was observed but up to 80% of the cells
survived even at very high concentrations of fumagillin. Treating the
cells with cis-platin alone (bottom panel) resulted in an IC50 of
approximately 100 μM cis-platin. The presence of 0.1 μg/ml
fumagillin in addition to increasing levels of cis-platin reduced the
IC50 to approximately 50 μM. However, treating the cells with
cis-platin in the presence of 10 μg/ml fumagillin resulted in enhanced
cell survival with an IC50 of approximately 200 μM.

[0211]Regardless of the length of incubation time, there was an
enhancement of the effect of cis-platin in the presence of 0.1 μg/ml
fumagillin but the opposite effect when the cells were treated with
cis-platin in the presence of 10 μg/ml fumagillin (FIGS. 41 and 42).
These observations suggested that at low levels of fumagillin, a
favorable balance is achieved and the drug acts synergistically with
cis-platin leading to the death of more cells.

[0212]Three siRNAs were designed to target different regions of the
MetAP-2 message (FIG. 43), to determine the effect of inhibiting MetAP-2
expression. The MetAp-2 siRNAs were:

[0213]FIG. 44 shows the effect of siRNA #1 on the levels of MetAP-2
expression in OVCA 429. Control cells were transfected with the sense
strand of the same siRNA alone or treated with the transfection agent
(siPort lipid). Transfection of the cells with siRNA #1 reduced the
levels of MetAP-2 expression by half, even though the transfection
efficiency was not 100%. Little effect was observed on the levels of
expression of GAPDH in those cells, indicating that gene expression was
not generally affected by this treatment. Furthermore, treating the cells
with siRNAs #2 and #3 did not result in a reduction in MetAP-2
expression.

[0214]The ability of OVCA 429 to resist cis-platin when the expression of
MetAP-2 was blocked was tested by treating OVCA 429 cells with siRNA #1,
the sense strand #1 alone, or siPort lipid alone. After 48 hours of
incubation with the siRNA the cells were exposed to varying
concentrations of cis-platin or the corresponding concentrations of its
solvent, DMSO, for 24 hours. The results of this experiment were
quantified and are shown in FIG. 45. The results indicated that the
presence of siRNA #1 reduced the IC50 of OVCA 429 from 25 μM to
approximately 3 μM. FIG. 46 shows a photograph of the 96-well plates
after performing the MTT assay.

[0215]Taken together the results indicated that MetAP-2 is a useful target
for therapeutic intervention in ovarian cancer.

Example 5

[0216]In vitro Testing of Calpain 2 and S100A10 as Therapeutic Targets and
Reduction of S100A11 Expression in OVCA 429 Cells

549728 (Calpain 2)

[0217]Three siRNAs were designed to target different regions of the
Calpain 2 message using methods described above. The Calpain 2 siRNAs
were:

[0218]Each siRNA was introduced into OVCA 429 cells as described above.
FIG. 53 shows the results of the knockdown experiments in the OVCA 429
cells using these sequences. Sequences #1 and #3 knocked down the gene
expression levels by about 50% using the protocol described above. In
addition, OVCA 429 cells comprising siRNA #3 were treated with various
concentrations of cis-platin. Usually, the ICso of OVCA 429 cells is
around 25 μM cis-platin after a 24 hour exposure to the drug. As shown
in FIG. 54, Calpain 2 siRNA #3 reduced the ICso to 3.12 μM,
thereby increasing the sensitivity of the cells to cis-platin by several
fold. Also, OVCA 429 cells were treated with increasing concentrations of
Calpain inhibitor I (ALLN) in the presence or absence of cis-platin. As
shown in FIG. 55, the ALLN reduced the IC50 of these cells in the
presence of cis-platin to 12.5 μM. Thus, the Calpain 2 siRNA #3 had a
greater effect than ALLN on the sensitivity of the OVCA 429 cells to
cis-platin.

[0222]Each siRNA was introduced into OVCA 429 cells as described above.
FIG. 57 shows that siRNAs #1 and 2 knocked down gene expression levels in
the OVCA 429 cells by 50% and 25% respectively, using the methods
described above.

[0223]Commercially available matched sets of colon cDNAs were obtained
from BD Biosciences, Inc. (San Jose, Calif.) that were isolated from five
individuals obtained from non-tumor tissue and also from adjacent tumor
tissue.

[0224]Quantitative real-time PCR experiments were conducted to determine
the expression levels of MetAP-2, SPARC, 5100A10, 5100A11 and Calpain-2
in the normal and tumor colon tissues. FIG. 58 shows the expression
levels of MetAP-2 in 5 pairs of matched colon cDNAs. The data indicates
that two of the patients had highly elevated expression levels in tumor
cDNA compared to their matched non-tumor cDNA (patients B and C; FIG.
58). One other patient showed only slightly elevated expression in the
tumor cDNA compared to its matched non-tumor cDNA (patient A; FIG. 58).
The level of expression in OVCA 429 was used as a reference. Previous
reports have shown that hepatic metastasis of human colon cancer can be
prevented by the MetAP-2 inhibitor TNP-470 (Tanaka et al., 1995, Cancer
Res. 55:836-9). FIG. 59 shows that expression levels of SPARC mRNA were
elevated in 4 out of 5 matched tumor samples compared to their matched
non-tumor cDNAs. FIG. 60 shows that expression levels of S100A11 mRNA
were elevated in all of the matched tumor samples compared to their
matched non-tumor cDNAs. FIG. 61 shows that expression levels of S100A10
mRNA were elevated in 4 out of 5 matched tumor samples compared to their
matched non-tumor cDNAs. FIG. 62 shows that expression levels of
Calpain-2 mRNA were elevated in all of the matched tumor samples compared
to their matched non-tumor cDNAs.

[0225]Taken together these observations suggest that MetAP-2, as well as
SPARC, S100A11, S100A10, and Calpain-2, are therapeutic targets for colon
cancer patients.

Example 7

Sandwich ELISA for Detecting Secreted Proteins in Serum

[0226]The wells of 96-well microtiter plates are coated with antibodies
raised against a gene product of interest. Aliquots of the purified
recombinant target gene product are diluted serially and are used to
generate a standard curve for quantitation. Aliquots of patient sera are
then added to each well. The plate is covered to minimize evaporation and
is incubated at 4° C. for a few hours to overnight. The antigen is
removed and the wells are washed 3 times with phosphate buffered saline
(PBS). 300 μl of blocking solution (3% w/v fish gel solution in PBS)
is added to each well and incubated for 2 hours at room temperature.
Blocking solution is removed and the wells are washed 3 times with PBS.
The appropriate antibody conjugated to horseradish peroxidase is then
added to each well (100 μl per well) and incubated at room temperature
for 1-2 hours. The antibody is then removed and the wells are washed 3
times with NP-40 solution (0.05% v/v NP-40 in PBS). Binding is detected
by adding ABTS (Rockland Immunochemicals) to each well (at 100 μl per
well) for 30 minutes at room temperature and reading absorbance at 405 nm
using a microplate reader. If alkaline phosphate conjugates are used
instead of peroxidase, pNPP (Rockland Immunochemicals) is used instead of
ABTS to detect binding.

[0227]Once the limit of detection is determined from standard curves
generated using purified proteins, a number of subjects who do not have
cancer and a number of patients who have been diagnosed as having cancer
or benign conditions are tested to determine an expected range of
concentrations for the particular gene product of interest. The expected
range for patients with cancer defines the limit that can be used to
identify or distinguish a patient with ovarian cancer or a patient with
recurring disease from a responding patient or a healthy subject without
cancer.

Example 8

[0228]siRNA-Mediated "Knockdown" of Gene Expression:

[0229]siRNAs specific for several genes were tested for their ability to
reduce (or "knockdown") their respective genes in ovarian cancer cell
lines following the protocols as described above. In each case several
siRNAs were tested against a control (non-specific) siRNA that was
GC-content matched (from Dharmacon, Inc, Lafayette, Colo.) to the test
siRNAs. In some cases a negative control (no treatment) was also
included. The level of expression knockdown varied with different siRNAs.
The specific genes and the siRNA sequences for each specific gene are
described below:

[0235]All three sequences were successful in reducing the expression of
Fused toes, with sequence #2 giving a 43% reduction in the level of
mRNA.7. Three siRNAs were Generated for Grancalcin (BC005214):

[0236]All three sequences were successful in reducing the expression of
Grancalcin, with sequence #2 giving an 83% reduction in the level of
mRNA.8. Three siRNAs were Generated for SRB1/CLA1/CD3611 (BCO22087):

[0237]Target sequences #s 1 and 3 were successful in reducing the
expression of SRB1, with sequence #1 giving a 60% reduction in the level
of mRNA.9. Three siRNAs were Generated for KIAA0082 (BCO31890):

[0239]Sequence #1 gave a 57% reduction in the level of mRNA, sequence
#2 gave a 54% reduction, and sequence #3 gave a 43% reduction in the
level of mRNA.11. Three siRNAs were Generated for Calponin 2 (D83735):

[0243]Both sequences were successful in reducing the expression of HMT1
mRNA with sequence #1 giving approximately 70% reduction and sequence #2
just over a 60% reduction.15. Three siRNAs were Generated for MPP10
(X98494) and Tested in Hey Cells:

[0244]All three sequences gave a reduction in mRNA expression with
sequence #1 giving almost 90% reduction, and sequences #2 and 3 with
approximately 30% and 40% reduction respectively.16. Two siRNAs were
Generated for IGFBP-7 (BC017201) and tested in OVCA 429.

[0251]Target sequences #1 and #2 did not reduce mRNA levels compared with
control while #3 reduced mRNA by approximately 45% compared to control.

Example 9

[0252]In vitro Functional Testing of Validated Genes:

[0253]The ability of specific siRNAs to enhance the sensitivity of OVCA
429 and OVCAR-3 cells to cis-platin was examined substantially as
disclosed above in Examples 3-5 above. In each case cis-platin
sensitivity was enhanced in the presence of the specific siRNAs but not
with the non-specific (control) siRNA or the negative control (no
treatment). The data indicate that the genes tested may be functionally
involved in the development of cis-platin resistance in ovarian cancer
cell lines. The results are summarized in Table 4.

[0255]The following protocol was developed for examining tumor growth
using

[0256]OVCAR-3 cells and nude mice: OVCAR-3 cells (15 million/injection)
were inoculated under upper arm region of nude mice. Visible lumps
appeared after 25 days. The tumors were measurable at about 35 days after
inoculation and animals were then either treated with cis-platin at 4
μg/kg body weight administered by IP injection 3 times a week for 2
weeks, followed by 1 week with no treatment or treated with saline
solution alone as control. FIG. 63 shows the volume of the tumor as a
function of body weight of the two mice. The data demonstrated that the
control animal carrying the tumor continued to grow the tumor in the
absence of any chemotherapy (mouse #1). The animal that received the
cis-platin treatment, in contrast, exhibited a stabilization of the tumor
size (mouse #2). A photograph of the tumors before and after cis-platin
treatment is also shown.

[0258]Calpain 2 and S100A11 and the effects of siRNA expression as
measured by real time quantitative PCR are shown in FIG. 64. Fifteen mice
are split into three groups of five. One group is treated as control, is
injected with OVCAR-3 cells without siRNA expression, the second group is
injected with siRNA-expressing OVCAR-3 cells and the third group is
injected with GFP-specific siRNA expressing OVCAR-3 cells. After
measurable tumors become apparent, the control group receives a saline
injection, while the second and third groups receives the standard
cis-platin treatment. Tumor growth is observed as a function of body
weight as described above.

[0259]In another experiment, fifteen mice split into groups of five ads
inoculated with unadulterated (i.e., non-recombinant) OVCAR-3 cells and
tumors permitted to grow. One group is treated as control. After
measurable tumors become apparent, the control group receives a saline
injection, the second group receives the standard cis-platin treatment as
described above, and the third group receives the standard cis-platin
treatment combined with TNP-470 (a clinically-recognized fumagillin
derivative). Tumor growth is observed as a function of body weight as
described above.

[0260]The information disclosed in the Examples can be summarized as
follows:

[0261]It should be understood that the foregoing disclosure emphasizes
certain specific embodiments of the invention and that all modifications
or alternatives equivalent thereto are within the spirit and scope of the
invention as set forth in the appended claims.